Apparatus for magnetic separation of cells

ABSTRACT

Described here is an automated robotic device that isolates circulating tumor cells (CTCs) or other biological structures with extremely high purity. The device uses powerful magnetic rods covered in removable plastic sleeves. These rods sweep through blood samples, capturing, e.g., cancer cells labeled with antibodies linked to magnetically responsive particles such as superparamagnetic beads. Upon completion of the capturing protocol, the magnetic rods undergo several rounds of washing, thereby removing all contaminating blood cells. The captured target cells are released into a final capture solution by removing the magnetic rods from the sleeves. Additionally, cells captured by this device show no reduced viability when cultured after capture. Cells are captured in a state suitable for genetic analysis. Also disclosed are methods for single cell analysis. Being robotic allows the device to be operated with high throughput.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. patent application Ser. No.12/333,213, filed on Dec. 11, 2008, and from U.S. Provisional PatentApplication No. 61/013,238, filed on Dec. 12, 2007, both of which arehereby incorporated by reference in their entirety.

FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under contract HG000205awarded by the National Institutes of Health. The Government has certainrights in this invention.

REFERENCE TO SEQUENCE LISTING, COMPUTER PROGRAM, OR COMPACT DISK

None

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of cell isolation, includingthe isolation of cells from peripheral circulation, labeling cells ofinterest magnetically, and using an automatable apparatus to immobilizeand isolate viable labeled cells for further testing and culture.

2. Related Art

Background

Utilizing current technologies, it is possible to count the number ofcirculating tumor cells present in blood from breast cancer patients andpredict disease outcome [1,2]. However, due to the lack of additionalinformation about the population of circulating tumor cells, currentmethods do not offer insights into directing treatment or developingnovel therapies. Current targeted treatments based on breast cancersubtypes, e.g., Her2/neu or estrogen receptor (ER) status, only focus onthe subtype of the primary tumor [3,4,5]. Recent studies have shown thata portion of breast cancer metastases have different Her2 and ER statuscompared to the original primary tumors [6,7,8,9]. By analyzing thecirculating tumor cells (CTCs) for their genetic characteristics, onecan target treatment not only to the primary tumor, but also to cellsthat may contribute to and serve as surrogate markers of metastases,thus improving survival in women with metastatic disease.

CTCs can be collected through a relatively non-invasive blood draw.However, isolating, purifying, and characterizing these cells has provenchallenging. Several current technologies allow for isolation andcounting of circulating tumor cells from patient blood samples.Additionally, the expression of two or three biomarkers can also beassessed. However, while the CTCs are enriched during these protocols,they are often heavily contaminated with blood cells, may not be viable,or the RNA may be severely compromised, making it difficult to reliablymeasure gene expression or simultaneously measure the expression oflarge numbers of genes. Described below is a robotic device which allowsone to obtain completely purified, living CTCs. In combination withmultiplex qRT-PCR, genes from single CTCs isolated from the blood ofpatients with metastatic breast cancer were analyzed for expressionlevels. Thus, current techniques partially purify CTCs from blood, butthere is still residual contamination with other blood cells. Sometechniques also fix and permeabilize the CTCs, making them unsuitablefor downstream microarray analysis or in vitro and in vivo biologicalstudies.

CTC biology is still poorly defined because most studies assess only CTCburden. The characterization of CTCs is a nascent field; isolation ofCTCs specifically for multigene molecular analyses, in contradistinctionto counting, is challenging for multiple biological and technicalreasons. The cells are fragile, likely a result of mechanical stresseson CTCs in the blood stream and chemical effects of cytotoxicchemotherapy. Moreover, CTCs are extremely rare. For example, 81% ofmetastatic breast cancer patients will have less than ten CTCs in a 7.5cc tube of blood containing about 10¹⁰ blood cells. Technical factorsthat impede CTC gene expression analysis in currently availableplatforms include cell fixation and permeabilization, immobilization,and, most importantly, blood cell contamination. CTC fixation andpermeabilization performed prior to fluorescent labeling of CTCs canstructurally modify RNA and impact cell viability. CTC immobilization onsubstrates such as glass slides, filters, or microposts limit singlecell manipulation. Finally, even after enrichment, CTCs may becontaminated by thousands of leukocytes (white blood cells, WBCs) thatconfound expression analysis, requiring bioinformatic techniques tosubtract non-CTC gene expression. Thus, direct simultaneous analysis ofmany gene targets in single human CTCs has yet to be performed.

Certain commercial technologies use immunomagnetic enrichment.Commercial products include the CellTracks® AutoPrep® System andCellSearch™ Circulating Tumor Cell Kit (Immunicon Corporation,Huntingdon Valley, Pa.), MACS® separation technology (MiltenyiCorporation, Bergisch Gladbach, Germany), and the RoboSep® automatedcell separator (StemCell Technologies, Vancouver, Canada). With thesetechniques, the epithelial cells in the blood are labeled with magneticparticles attached to an antibody targeted to an epithelial cell surfacemarker, usually EpCAM. The blood is processed and external magnets holdthe epithelial cells at the side of the tube, while the other bloodcells are diluted and pipetted out or eluted through a column. Theremaining epithelial cells are then available for immunocytochemicalanalysis, again amidst 1000-10,000 WBCs. Because of heavy mononuclearcell contamination (whose nucleic acids or proteins would overwhelm anysubsequent molecular analyses of the CTCs), most analyses stain andcount cells, or limit characterization to one to two immunostains.

Specific Patents and Publications

U.S. Pat. No. 3,970,518 to Giaever, issued Jul. 20, 1976, entitled“Magnetic separation of biological particles,” discloses a method andapparatus in which the particular cell population that is to beseparated from a mixed population is contacted with small magneticparticles or spheres which are first provided with a monomolecularcoating of antibody to this select population. As the metallic particlesenter the field created by a coil at the bottom of the vessel, they arecaptured and immobilized while liquid is unaffected and leaves vessel.

U.S. Pat. No. 7,125,964 to Luxembourg, et al., issued Oct. 24, 2006,entitled “Purification of antigen-specific T cells,” discloses a methodto capture, purify and expand antigen-specific T lymphocytes, usingmagnetic beads coated with recombinant MHC class I molecules. Theinventors used attachment of biotinylated MHC Class I molecules onAaidin-coated magnetic beads.

U.S. Pat. No. 5,200,084 to Liberti, et al. Apr. 6, 1993, entitled“Apparatus and methods for magnetic separation,” discloses a magneticseparation apparatus and methods for separating colloidal magneticparticles from a non-magnetic test medium in which the magneticparticles are suspended. The separator comprises a container holding thenon-magnetic test medium, one or more magnetic wires disposedsubstantially within the test medium in the container and an externalmagnet (illustrated at 31 of FIG. 1 of the patent) for producing amagnetic field gradient within the test medium. According to the methodof the invention, the container holding the test medium is positioned inthe separator, producing a magnetic field gradient operative to causethe magnetic particles to be attracted to the areas surrounding themagnetized wires and to adhere to the wires.

U.S. Pat. No. 5,837,144 to Bienhaus, et al., issued Nov. 17, 1998,entitled “Method of magnetically separating liquid components,”discloses Method of separating a component of a liquid from othercomponents by immobilizing the component to suspended magnetic particlesin a vessel, immersing a magnetic device into the vessel while thedevice is separated from the liquid by means of a protective sleeve madeof a non-magnetic material. The protective sleeve is selected such thatits outer surface is always spaced apart from the inner surface of thevessel by approximately the same distance.

U.S. Pat. No. 6,468,810 to Korpela, issued Oct. 22, 2002, entitled“Magnetic particle transfer device and method,” discloses a pipette likedevice for transfer suitable for capturing and releasing microparticlesbinding an immobilized substance, which includes a magnet as well aseither an extendable membrane, shapable membrane or magnet's coatingsuch that the membrane or coating pressing tightly against the magnet'ssurface separates the magnet from the microparticles but does notsubstantially weaken the magnetic field directed at the microparticles(See FIGS. 1D and 1E of the patent).

US 20070251885 by Korpela et al., published Nov. 1, 2007, entitled“Method and a Device for Treating Microparticles,” disclose a method forhandling microparticles in such a manner, that at least two treatmentsteps are performed for microparticles in the same vessel without movingthe particles to another vessel. This can be brought about by moving themagnet inside the ferromagnetic tube in such a manner, that it can becompletely inside the tube, whereupon the efficiency of the magnet isinsignificant or nonexistent, or it can be partially or completelyoutside the tube, whereupon the efficiency and the collecting area ofthe magnet are in relation to the protruding part of the magnet.

BRIEF SUMMARY OF THE INVENTION

The following brief summary is not intended to include all features andaspects of the present invention, nor does it imply that the inventionmust include all features and aspects discussed in this summary.

In certain aspects, the present invention comprises a method forcapturing and isolation of cells comprising: (a) mixing a samplecomprising rare target cells and contaminant cells with magneticallyresponsive particles having an affinity to the rare target cells toproduce a solution having magnetically responsive particles bound torare target cells; (b) contacting a magnetic member having anon-adherent sleeve with the solution; (c) producing a continuousrelative motion between the magnetic member and the solution while themagnetic member produces a magnetic field across the non-adherentsleeve, such that the magnetically responsive particles bound to raretarget cells are selectively captured onto the non-adherent sleeve; (d)contacting the magnetic member having the captured rare target cellswith a recovery solution; (e) substantially removing the magnetic fieldproduced by the member across the non-adherent sleeve such that themagnetically responsive particles bound to rare target cells arereleased into the recovery fluid.

In certain aspects, the present invention comprises a device for captureand isolation of target cells from a mixed cell population wherein thetarget cells are labeled with magnetically responsive material to formlabeled target cells, where the device comprises (a) magnetic memberhaving a tip and another end connected to an actuator; (b) a sleevebetween the magnetic member and the mixed cell population to preventdirect contact of cells with the magnetic member, said sleeve beingnonmagnetic and nonadherent to cells in the mixed cell population; (c)said sleeve being separable from the magnetic member by the actuator,which causes movement between the magnetic member and the sleeve,whereby the magnetic member is moved more or less into the sleeve; and(d) said actuator being constructed and arranged for causing in thedevice (i) relative stirring movement between the magnetic member andthe mixed cell population to contact the magnetic member with a majorityof cells in the mixed cell population, and (ii) causing movement of themagnetic member into and out of the container to retrieve target cellsfrom the mixed cell population. The term “sleeve” as used here, refersto a covering that can be fitted to and closed to completely cover aregion of the magnetic member that is in contact with the cells. Therelative stifling movement, movement into and out of the sleeve and intoand out of container(s) is preferably accomplished by mounting theactuator on a robotic mechanism for x-y-z movement, such roboticactuators being known in the art for other purposes.

The magnetic member in (a) may be a rod having a diameter of at least 4mm to produce a high magnetic field strength and having a tapered tipfor producing a high magnetic gradient. The magnetic gradient at the tipmay be used to concentrate the magnetic field in a smaller area near thetip, which is on the container in use and contacting the target cells.It has been found that the magnetic member may advantageously have atapered tip. This may be rounded or of a compound shape. In furtheraspects, the magnetic member possesses a field strength at the tip of atleast 0.2 to 1 Tesla, or about 0.5 Tesla. Measured another way, themagnetic member may have a pull strength of at least 70 pounds. Incertain aspects, the device comprises the use of a rare earth magnet.The magnetic member may thus comprise neodymium, iron and boron (NIBmagnet). The sleeve material is nonadherent, and may consist essentiallyof a material selected from the group consisting of vinyl polymer,paramagnetic metal, ceramic material, and polyHEMA. The vinyl polymermay be medical grade PVC. To accomplish efficient sweeping, the actuatormay cause orbital movement to contact the magnetic member with amajority of cells. Orbital movement may be, for example, outwardlyexpanding concentric circles, expanding outward until near the wall(s)of the container. The device may comprise (and in use may use) one ormore containers for holding the sample, for holding a wash solution andfor holding target cells after isolation from the mixed cell population.In certain embodiments, there are separate containers for holdingsample, wash solution and target cells. Like the sleeve, the containermay be designed to comprise a material which is non-adherent to targetcells. Rather than a container that closely approaches the magneticmember, the container may define an open pool for holding the sample.The pool will have a surface area considerably larger than the crosssectional area of the magnetic member and will have no external meansfor movement of the sample during sweeping. This facilitates thestifling, or sweeping. The actuator may comprise a component whichcauses the magnetic member to sweep through the pool in concentriccircles. There may, in certain embodiments, only one magnetic member, oran array, with one magnetic member in each container. The device maycomprise multiple magnetic members controlled by a single actuator.

The device may further comprise apparatus for further processingpurified target cells left in the wash solution. The device may comprisea probe for extracting a single target cell from a collection of targetcells isolated after removal of contaminant cells. The furtherprocessing apparatus may comprise a computer programmed with imagerecognition software and controlling the probe. The probe may bedirected towards a concentration of target cells obtained when a magnetis used to concentrate the cells in a recovery or wash container.

In certain aspects, the present invention comprises a method. Itcomprises a method for capture and isolation of intact, rare targetcells in a sample having a mixed cell population of target cells andcontaminant cells, wherein the target cells are labeled withmagnetically responsive material to form labeled target cells,comprising: (a) labeling the sample with a label specific for targetcells, said label being magnetically responsive, to form a labeledsample; (b) contacting the labeled sample with a member extending intothe sample and comprising a strong magnet covered by a nonmagnetic,nonadherent sleeve; (c) sweeping the member through the sample to causecells to attach to the sleeve; (d) washing the sleeve with cellsattached to remove unlabeled cells; (e) separating the magnet from thesleeve, whereby labeled cells are removed from the sleeve; and (f)collecting labeled cells to form a composition comprising beads andlabeled cells. The term “washing” may include either or both of simplyimmersing the bound cells on the rod into a cell free solution, or astep of moving the cells attached to the sleeve into a cell freesolution, releasing the bound cells and recapturing them. The releaseand recapture step has been used in the presently exemplified methods.

The present methods may further include further processing of anisolated cell (e.g., CTC). They may include the step of isolating asingle labeled cell from the beads (or a single bead). The methods mayinclude the use of certain cell surface markers, such as an HLA marker(e.g., HLA-A2), an epithelial cell surface marker, such as EpCAM, or astem cell marker such as CD44 or CD96.

As described below, the method does not require pre-processing and maybe carried out. It may, in certain method embodiments, the sample ishuman peripheral blood which has only been minimally treated, such as bydilution and anticoagulation. As discussed below, the target cells maybe rare cells such as CTCs. The method of the present invention maycomprise in certain aspects, multiple washing steps as recited in step(d). The method of the present invention may comprise in certainaspects, the step of extracting intact genetic material from isolatedtarget cells. This may further comprise the step of testing forexpression of certain genes in extracted genetic material. The analysisof the genetic material may be useful in the study of individual CTCsfrom a given patient. The genes to be analyzed for expression level mayinclude GAPDH, beta actin, big ribosome protein (RPLPO) (the foregoingbeing housekeeping genes), CRYAB, EGFR, FOXA1, CD44, ESR1 (ER) PGR (PR),and mutated forms of Myc, Ras, BRCA1, BRCA2, APC, and p53. The method ofthe present invention may comprise in certain aspects, testing for theexpression level of the vimentin gene, where increased expression ofvimentin indicates a putative mesenchymal CTC. The present device andmethods enable the preparation of a relatively large number of purifiedCTCs. In certain aspects, the present invention may comprise an isolatedpopulation of viable CTCs having at least 10 cells of at least 90%purity of CTCs. For example, in a population of 100 cells, about atleast 90 of them will be CTCs. As noted, the CTCs of the presentisolation method are viable and do not have altered gene expressionpatterns. The presently exemplified compositions are CTCs which arebreast cancer cells. It has also been found that the presently preparedcompositions comprise a putative MSC (mesenchymal-like cancer stemcell).

One aspect of the invention comprises a method for capturing andisolating cells comprising: (a) mixing a sample comprising rare targetcells and contaminant cells with magnetically responsive particleshaving an affinity to the rare target cells to produce a sample solutionhaving magnetically responsive particles bound to rare target cells; (b)contacting a magnetic member having a non-adherent sleeve with thesample solution; (c) producing a continuous relative motion between themagnetic member and the sample solution while the magnetic memberproduces a magnetic field across the non-adherent sleeve, such that themagnetically responsive particles bound to rare target cells areselectively captured onto the non-adherent sleeve; (d) contacting themagnetic member having the captured rare target cells with a recoverysolution; (e) substantially removing the magnetic field produced by themember across the non-adherent sleeve whereby the magneticallyresponsive particles bound to rare target cells are released into therecovery fluid.

In some embodiments, the non-adherent sleeve comprises a sleeve over themagnetic member, and step (e) of substantially removing magnetic fieldacross the non-adherent sleeve comprises removing the magnetic memberfrom at least a portion of the sleeve.

In some embodiments, the magnetic member comprises an electromagnet, andstep (e) of substantially removing the magnetic field across thenon-adherent sleeve comprises demagnetizing the electromagnet.

In some, alternative, embodiments, the magnetic member is heldstationary in a fluid flow channel and the continuous relative motionbetween the magnetic member and the sample solution is produced byflowing the sample solution past the magnetic member.

In certain aspects of the invention, the continuous relative motionbetween the magnetic member and the sample solution produces a velocityin the non-turbulent flow regime. In some embodiments, the continuousrelative motion between the magnetic member and the sample solution hasa Reynolds number of 0.1 to 100. In some embodiments, the continuousrelative motion between the magnetic member and the sample solution hasvelocity of 0.1 mm/sec to 1 mm/sec. In some embodiments, the magneticmember has a surface field strength of 0.2 Tesla to 1 Tesla. In someembodiments, the magnetic member has a surface field strength of 0.2Tesla to 1 Tesla and the continuous relative motion between the magneticmember and the sample solution has velocity of 0.1 mm/sec to 1 mm/sec.

In some embodiments, the continuous relative motion is applied such thata majority of the sample solution has access to the magnetic member.

In some embodiments, the non-adherent sleeve comprises a polymerselected from the group consisting of vinyl, chlorofluorocarbon polymersand silicone. In some embodiments, the non-adherent sleeve comprisespolyvinyl chloride (PVC).

In some embodiments, the method further comprises contacting themagnetic member having the captured rare target cells with a wash fluidafter step (c).

One aspect of the invention comprises a method for capturing andisolation of cells comprising: (a) contacting a magnetic member having anon-adherent sleeve with a sample solution comprising rare target cellsand contaminant cells wherein the rare cells constitute less that 0.1%of the cells in the sample and wherein some or all of the rare targetcells are bound to magnetically responsive particles; (b) moving themagnetic member relative to the sample solution to capture rare targetcells bound to magnetically responsive particles; and (c) releasing therare target cells from the non-adherent sleeve into a recovery solution,whereby the capture rate is greater 35%, and the purity is greater than99%.

In some embodiments, the sample solution is blood and the rare cells arepresent in the sample solution at less than 1 in 50 million, and thecapture rate is greater than 75% and the purity is greater than 90%. Insome embodiments, the sample solution is blood and the rare cells arepresent in the sample solution at less than 1 in 500 million, and thecapture rate is greater than 35% and the purity is greater than 25%. Insome embodiments, greater than 80% of the recovered rare target cellsare intact. In some embodiments, greater than 80% of the recovered raretarget cells are viable.

One aspect of the invention comprises a device for capturing andisolating cells comprising: (a) a sample vessel for holding a samplesolution comprising rare target cells and contaminant cells wherein someor all of the rare target cells are bound to magnetically responsiveparticles; (b) a magnetic member having a non-adherent sleeve; (c) arecovery vessel for holding a recovery solution; (d) an actuator forcausing continuous relative movement of the magnetic member (i) betweencontainers, (ii) into and out of the vessels, and (iii) at least twodirections within the sample vessel to provide continuous relativemotion between the magnetic member and the sample solution to provideselective capture of magnetically responsive particles having bound rarecells; and (e) a mechanism for producing a magnetic field across thenon-adherent sleeve while the magnetic member is in the sample solution,and for substantially removing the magnetic field across thenon-adherent sleeve while the non-adherent sleeve is in the recoverysolution. The sample vessel may be configured to hold the sample as apool, as described above.

In some embodiments, the non-adherent sleeve comprises a sleeve over themagnetic member, and the mechanism for substantially removing themagnetic field across the non-adherent sleeve comprises moving themagnetic member out of at least a portion of the sleeve.

In some embodiments, the magnetic member comprises an electromagnet, andthe mechanism for substantially removing the magnetic field across thenon-adherent sleeve comprises demagnetizing the magnetic member.

One aspect of the invention comprises a device for capture and isolationof target cells from a mixed cell population wherein the target cellsare labeled with magnetically responsive material to form labeled targetcells, comprising: (a) a magnetic member having a tip and another endconnected to an actuator; (b) a sleeve between the magnetic member andthe mixed cell population to prevent direct contact of cells with themagnetic member, said sleeve being nonmagnetic and non-adherent to cellsin the mixed cell population; (c) said sleeve being separable from themagnetic member by the actuator, which causes movement between themagnetic member and the sleeve, whereby the magnetic member is movedmore or less into the sleeve; and (d) said actuator being constructedand arranged for causing in the device (i) relative stirring movementbetween the magnetic member and the mixed cell population to contact themagnetic member with a majority of cells in the mixed cell population,and (ii) causing movement of the magnetic member into and out of thecontainer to retrieve target cells from the mixed cell population.

In some embodiments, the magnetic member is a rod having a diameter of 1mm to 10 mm to produce a high magnetic field strength and having atapered tip for producing a high magnetic gradient. In some embodiments,the magnetic member has a tapered tip. In some embodiments, the magneticmember possesses a field strength at the tip of at least 0.5 Tesla. Insome embodiments, the magnetic member is a rare earth magnet. In someembodiments, the magnetic member has a pull strength of at least 70pounds. In some embodiments, the magnetic member has a surface fieldstrength of 0.2 to 1 Tesla. In some embodiments, the magnetic membercomprises neodymium, iron and boron.

In some embodiments, the sleeve consists essentially of a materialselected from the group consisting of vinyl polymer, paramagnetic metal,ceramic material, and polyHEMA.

In some embodiments, the magnetic member consists essentially ofneodymium, iron and boron alloy.

In some embodiments, the actuator causes orbital movement to contact themagnetic member with a majority of cells.

In some embodiments, the device further comprises one or more containersfor holding the sample, for holding a wash solution and for holdingtarget cells after isolation from the mixed cell population. In someembodiments, there are separate containers for holding sample, washsolution and target cells. In some embodiments, the container comprisesa material which is non-adherent to target cells.

One aspect of the invention comprises a device for capture and isolationof target cells in a sample having a mixed cell population of targetcells and contaminant cells, wherein the target cells are labeled withmagnetically responsive material to form labeled target cells,comprising: (a) a container for holding the sample; (b) a magneticmember having a tip; (c) a sleeve between the magnetic member and themixed cell population to prevent direct contact of cells with themagnetic member, said sleeve being nonmagnetic and non-adherent to cellsin the mixed cell population; (d) said sleeve being separable from themagnetic member by an that actuator causes movement between the magneticmember and the sleeve whereby the magnetic member is moved more or lessinto the sleeve; and (e) said actuator being constructed and arrangedfor causing (i) relative stifling movement in the container between themagnetic member and the mixed cell population to contact the magneticmember with a majority of cells in the mixed cell population, and (ii)causing movement of the magnetic member into and out of the container toretrieve target cells from the mixed cell population.

In some embodiments, the device further comprises a container whichcomprises a portion having a magnet opposite the magnetic member, forattracting isolated labeled target cells from the magnetic member informing an isolated population. In some embodiments, the containerdefines an open pool for holding the sample. In some embodiments, theactuator comprises a component which causes the magnetic member to sweepthrough the pool in concentric circles. In some embodiments, the devicecomprises only one magnetic member in one container. In someembodiments, the device comprises multiple magnetic members controlledby a single actuator.

In some embodiments, the device further comprises a probe for extractinga single target cell from a collection of target cells isolated afterremoval of contaminant cells. In some embodiments, the device comprisesa computer programmed with image recognition software and controllingthe probe. In some embodiments, device further comprises a magnet forremoving target cells from the sleeve.

One aspect of the invention comprises a method for capture and isolationof intact, rare target cells in a sample having a mixed cell populationof target cells and contaminant cells, wherein the target cells arelabeled with magnetically responsive material to form labeled targetcells, comprising: (a) labeling the sample with a label specific fortarget cells, said label being magnetically responsive, to form alabeled sample; (b) contacting the labeled sample with a memberextending into the sample and comprising a strong magnet covered by anonmagnetic, non-adherent sleeve; (c) sweeping the member through thesample to cause cells to attach to the sleeve; (d) washing the sleevewith cells attached to remove unlabeled cells; (e) separating the magnetfrom the sleeve, whereby labeled cells are removed from the sleeve; and(f) collecting labeled cells to form a composition comprising beads andlabeled cells.

In some embodiments, the method further comprises the step of isolatinga single labeled cell from the beads. In some embodiments, the label isan antibody against an cell surface marker selected from the groupconsisting of HLA, EpCAM, CD44 and CD 46. In some embodiments, thesample is human peripheral blood. In some embodiments, the target cellsare CTCs.

In some embodiments, a strong magnet is a rare earth magnet having anaxial dipole whereby magnetic particles are concentrated at oneelongated end of the magnet. In some embodiments, the non-adherentsleeve is a polymer. In some embodiments, the polymer is a polymerselected from the group consisting of vinyl, chlorofluorocarbon polymersor silicone.

In some embodiments, the method further comprises the step of applying amagnetic field opposite the sleeve after the magnet has been removed toassist in removing labeled cells from the sleeve.

In some embodiments, the method further comprises multiple washing stepsas recited in step (d) above.

In some embodiments, the method further comprises the step of extractingintact genetic material from isolated target cells. In some embodiments,method further comprises the step of testing for expression of certaingenes in extracted genetic material.

In some embodiments, genes are selected from the group consisting ofGAPDH, Beta Actin, Big ribosome protein (RPLPO), CRYAB, EGFR, FOXA1,CD44, ESR1 (ER), PGR (PR), and mutated forms of Myc, Ras, BRCA1, BRCA2,APC, and p53.

In some embodiments, the method comprises the testing for the expressionof vimentin, where increased expression of vimentin indicates amesenchymal CTC.

One aspect of the invention comprises a composition comprising anisolated population of viable CTCs having at least 1 cells of at least25% purity of CTCs. In some embodiments, the CTCs are breast cancercells. In some embodiments, composition further comprises an MSC orputative MSC.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-D are a series of four illustrations representing a schematicdiagram of one embodiment of a device according to the presentinvention, where magnetic members in the form of wires are used, where1A shows sweeping to pick up cells; 1B shows lifting of attached cellsfrom the medium; 1C shows deposition of removed cells from 1B into adifferent medium, and 1D shows separation of the cells from the wires.

FIG. 2A-D is a series of four photographs showing a device without asleeve (2A) after the wire has been inserted into a cell containingmedium and allowed to incubate and then removed (2B), cell capture witha sleeve-covered wire (2C) and a wire removed from a sleeve (2D),showing that the cells have fallen off; the white size bar in FIGS. 2Aand 2B is 100 microns (μM);

FIG. 3A-F is a series of six drawings which illustrate a schematicrepresentation of one embodiment of a device according to the presentinvention.

FIGS. 4A and 4B are photographs showing 40× magnification of MCF7 cells(expressing GFP) added to donor blood, labeled with Dynabeads® andcaptured with the device. FIG. 4A shows brightfield, and 4B shows afluorescent image of the same cells. Numerous beads surrounding twocells can be seen in FIG. 4A.

FIG. 5A-C are photographs showing 40× magnification of a selection ofCTCs, labeled with Dynabead® beads and isolated using the device, theCTCs being from two patients (SM014 and SUBL018). FIG. 5A is aSM014 cell7, 5B SUBL017 cell 18, and 5C SUBL017 cell 23. FIG. 5 shows numerousbeads attached to target cells.

FIG. 6A-E is a series of five graphs showing amplification plots showingthe integrity of cells isolated with the present device, with 15different genes as indicated, in samples containing (FIG. 6A), 0.1 ng ofhuman reference RNA; FIG. 6B a single MCF7 cultured cell; FIG. 6C SM014cell number 7; FIG. 6D SUBL017 cell 18; and FIG. 6E SUBL017 cell 23.

FIGS. 7A and B comprise drawing showing the design of an embodiment ofthe present device using magnetic rods and a plastic sleeve, where thereare six rods and six sleeves, and the sleeves are collectively joined toa sleeve holder, as shown in side view, FIG. 7A. As seen in top view(FIG. 7B), the sleeves and rods are arrayed so that each can bemanipulated and placed into a different sample container.

FIG. 8A-F is a series of sketches showing various magnet designs, shownfrom right to left as FIG. 8A, no magnet; FIG. 8B a single rod below thesupport plate; FIG. 8C, four sections below the plate; FIG. 8D, narrowsingle rod below the plate; FIG. 8E, six sections in a casing; and FIG.8F six sections without a casing. All magnets in these figures have twoshort sections above the support plate.

FIG. 9 is a drawing showing the magnetic profile of a magnetic rod in aplastic sleeve. On cells with attached magnetic beads (black circles),the magnetic rod produces a magnetic force in z proportional to the nonuniformity (dB²/dz) of the magnetic field, thus imparting momentum in az direction proportional to (dB²/dz) and to a dwell time that dependsboth on the sweep speed and on the velocity distribution across theboundary layer that extends into the fluid from the surface of thesleeve. On cells with no beads (open circles) only a transverse force inthe direction of sweeping (large arrow) acts on the cell. To capture alabeled cell, the transverse momentum must be sufficient to overcome thedrag of the fluid on the cell so that it reaches the sleeve surfacewithin the dwell time. Magnetic strength and sweep speed were controlledfor these parameters to obtain high capture efficiency at the tip of themagnetic member.

FIG. 10A-E are photomicrographs of CTCs from patients with metastaticbreast cancer. FIGS. 10A, B, C, D and E are 5 representativephotomicrographs of CTCs isolated from Patients A, B, D, E, and F,respectively.

FIG. 11A-C is a series of three graphs showing amplification curves forCD45 and β-actin in four individual CTCs (11A), leukocytes (11B), andReference RNA (11C). The four individual CTCs were isolated from twowomen with metastatic breast cancer, and 0.01 ng of human reference RNAwas used in 11C.

FIG. 12 is a cluster analysis of single CTCs normalized for expressionof GAPDH showing different populations of CTCs, including a putativemesenchymal-like cancer stem cell. Scale bar represents Δ Ct; higher ΔCt values indicate lower gene expression.

FIG. 13 is a schematic drawing of an automated cell extraction system,where clusters of cells may be manipulated to yield individual cells.

FIG. 14 is a schematic drawing showing an arrangement of a magnetdesigned to have a large gradient of magnetic field.

FIG. 15 is a schematic drawing showing an exemplary embodiment of theinvention comprising a fluidic channel.

FIG. 16A-D is a schematic drawing showing an alternative exemplaryembodiment of the invention comprising a fluidic channel and a magneticmember that can be inserted or removed from a hollow pillar; (A) showscells prior to attachment; (B) shows cells attached to the magnetic,sleeved roof; (C) shows a wash step with cells attached; and (D) showsrelease.

FIG. 17A-C is a schematic drawing illustrating assisted release with ahigh flow rate and assisted release with an external magnet in anembodiment comprising a fluidic channel; (A) shows wash step; (B) showsassisted release; and (C) shows assisted release with external magnet.

FIG. 18 is a schematic drawing showing a magnetic member inserted into afluidic channel.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. Generally, nomenclatures utilized inconnection with, and techniques of, cell and molecular biology andchemistry are those well known and commonly used in the art. Certainexperimental techniques, not specifically defined, are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification.

Overview of Magnetic Separation Devices and Methods

The devices described below allow one to isolate and purify rare cellssuch as CTCs by labeling them with magnetically responsive particles andthen uses one or more permanently magnetized or magnetizable memberssuch as wires or rods with non-adherent sleeves such as removablesleeves for several rounds of capture-and-release of cells. Analysis andpreparation of isolated CTCs is therefore enabled. Throughout theprocess, cell viability may be maintained (no fixation orpermeabilization steps), and all contaminating blood cells are removed.Thus this technology can isolate living circulating tumor cells (CTCs)from patients with metastatic disease for further studies, as well asisolating other rare cell populations. Rare cells generally are thosecomprising less than about 0.1% of a heterogeneous cell population.

The device may be used for capture and isolation of intact, rare andviable target cells (which may, in certain embodiments, includesubcellular components or particles) in a sample having a mixed cellpopulation of target cells and contaminant cells. The target cells maybe rare, comprising a very small fraction of the cellular population.For example, rare tumor or fetal or stem cells may be found in adulthuman peripheral blood. The method and device are also applicable toanimals, or other biological tissues, or cell cultures. The target cellsare labeled with magnetically responsive material, which may be aferromagnetic or paramagnetic bead, or an iron or gold nanoparticle, orthe like. A paramagnetic material includes, e.g., such as aluminum,magnesium, and platinum, and, as is known, refers to a material which isnot ferromagnetic, but is only weakly pulled towards a strong magnet.Ferromagnetic materials such as nickel, cobalt, pure iron, iron alloys,certain rare earths such as gadolinium, dysprosium) etc. are stronglyattracted to magnets and are preferred materials for the magneticallyresponsive particles.

The magnetically responsive particles (e.g., beads) are linked to aligand specific to the target cell, generally an antibody, but theligand may also comprise another cell specific ligand, such as a proteinor lipid. The choice of ligand can depend on the target cell to beisolated. The devices of the present invention are intended to beautomated and perform certain preprogrammed steps automatically andrepeatedly. In some embodiments, the technology comprises or is usedwith one or more containers, as the device carries out certain stepsinvolving thorough sample contact, washing off of contaminating cells,and collection of an enriched target cell population. The containers aredesigned in certain respects to allow sweeping through a relativelystill pool of sample, said sweeping covering a majority, orsubstantially all, of the surface of the pool. The device comprises amagnetic member constructed, for example, to obtain high magneticstrength. This may be a rare earth magnet or may be a material that ismagnetizable, provided that the required field strengths can beobtained. It may be an electromagnet that is capable of being magnetizedfor capture and de-magnetized for release. The magnetic member has, onits outer surface, in the portion that will contact the liquid, anon-adherent sleeve. In some embodiments, the magnet member is adaptedto fit closely into an thin, self-supporting sleeve surrounding themagnetic member to prevent direct contact of sample cells with themagnetic member, said sleeve being tightly fitted to, yet separablefrom, the magnetic member, said sleeve further being constructed of amaterial which is non-adherent to cells in the mixed cell population.

By “nonadherent” it is generally meant that the cells (at least 90%,preferably at least 99%) will easily wash off or fall off of the sleeve.The level of adherence of cells to a material such as a plastic can bemeasured by methods well known in the art. Generally, a material isexposed to a sample containing cells, then the material are exposed tofresh solution, and the number of cells that adhere to the material isdetermined. For instance, plastic pieces can be incubated in whole bloodat room temperature, for example for 30 minutes. The pieces are thenwashed with phosphate buffer solution (PBS). The number of cells adheredafter washing is determined to determine the adherence of the cells.Materials that are non-adherent generally have fewer than 1,000 cellsper square millimeter. Particularly useful materials will have evenfewer cells, for example less than 100 cells/mm² or less than 100cells/mm². We have found that polyvinylchloride, in particularbiological grade polyvinylchloride has less than 100 cells/mm².

In an alternative embodiment the non-adherent sleeve is not removed, butthe magnetic member is demagnetized in order to remove the magneticfield across the non-adherent sleeve to allow release of the cells. Insome embodiments, a second magnet is used to facilitate cell removalfrom the non-adherent sleeve. The second magnet can be, for example, amagnet disposed below the recovery vessel. The magnetically responsiveparticles, now released from the field of the magnetic member will beattracted to the second magnet, facilitating recovery of the particles.One preferred non-adherent material that can be used as a sleeve isbiological grade PVC, which has been found to be surprisinglynon-adherent. The non-adherent sleeve is generally nonmagnetic, and“magnetically transmissive,” meaning that it is of a material (such asthe exemplified organic polymer) and also of a dimension which allowsthe magnetic force of the magnet to pass through the sleeve with littleimpedance. To this end, the sleeves are generally constructed to bethin. It is generally contemplated that the sleeve will be on the orderof 1 to 1000 μm, in some embodiments, 10-300 μm thick, and in someembodiments, about 10-30 μm thick. The sleeve may be deformable so thatit changes shape to accommodate an inserted magnetic member, e.g., asshown in FIG. 18. When the magnet member (or members, there being thepossibility of numerous, parallel members having individual non-adherentsleeves) has been immersed in and swept through the sample to thoroughlyattract the rare cells, e.g., “stirred” through the sample, in someembodiments, a wash step is used in order to wash off the contaminatedcells. The wash step can be performed by spraying or rinsing themagnetic member with a wash solution. The wash step can also beperformed by immersing the magnetic member into a container having awash solution. To this end, there is provided a wash vessel or washcontainer for holding an immersing wash solution for removingcontaminant cells while target cells remain bound to the sleeve. Thewash vessel generally holds a volume of liquid sample which is sized forhigh fluid throughput. To this end, the container may be many times thecross sectional area of the magnet. That is, the magnet may be 0.6 cmdiameter, while the container(s) may be on the order of 20 to 50 timeslarger diameter. After one or more washings, there is provided in thedevice a recovery vessel or recovery container for holding target cellsisolated after removal of contaminant cells. To accomplish the abovemovements, and the fluid movement, in some embodiments, there isprovided an actuator for moving the magnetic member between containersand into and out of containers, whereby the magnetic member is immersedwithin the mixed cell population; into and out of the sleeve; and in atleast two directions within the sample for sweeping the sleeved memberthrough the sample.

The method and device described here can be used to isolate circulatingtumor cells (CTCs), and may be used in any case where rare cells orother biological structures can be magnetically labeled for laterseparation and isolation. The presently constructed device can process,for example, 9 ml blood/1 hr and captures more than 50% of CTCs asmeasured in spiking experiments. The below-described devices, includingan exemplified embodiment known as a MagSweeper, gently enrich targetcells such as CTCs by 10⁸-fold from blood. Purified cells can then beindividually selected for biochemical analysis. To this end, a devicefor probing cell clusters washed from a magnetic member is provided. Thetechnology described herein is capable of obtaining capture rates andlevels of purity of rare cells obtained from mixed cell populations wellabove those of other technologies. Capture rate generally refers to thepercentage of target rare cells that are captured relative to the targetrare cells in the sample. Purity generally refers to the percentage ofrare cells over the total number of cells in the final recoveredsolution, i.e., (number of rare cells)/(number of rare cells plus numberof contaminant cells) in the recovered solution. In some embodiments,the technology can achieve 99.9%, 99.99% or 100% purity from a samplewith a mixed cell population, e.g., a blood cell population of about 1in 5 million or greater. In some embodiments, the technology can achieve90%, 99% or 99.9% purity from a sample with a mixed cell population,e.g., a blood cell population of about 1 in 50 million or greater. Insome embodiments, the technology can achieve 90%, 99% or 99.9% purityfrom a sample with a mixed cell population, e.g., a blood cellpopulation of about 1 in 50 million or greater. In some embodiments, thetechnology can achieve 25%, 90% or 98% purity from a sample with a mixedcell population, e.g., a blood cell population of about 1 in 500 millionor greater.

In some embodiments, the technology can achieve capture rates of 35%,from a sample with a mixed cell population, e.g., a blood cellpopulation of about 1 in 5 million, about 1 in 50 million, or about 1 in500 million. In some embodiments, the technology can achieve capturerates of 75%, from a sample with a mixed cell population, e.g., a bloodcell population of about 1 in 5 million, about 1 in 50 million, or about1 in 500 million. In some embodiments, the technology can achievecapture rates of 90%, from a sample with a mixed cell population, e.g.,a blood cell population of about 1 in 5 million, about 1 in 50 million,or about 1 in 500 million.

In some embodiments, the technology can achieve a combination of capturerate and purity above what is achieved with current methods. In someembodiments, the technology can achieve a 35% capture rate and 99.9%,99.99% or 100% purity from a sample with a mixed cell population, e.g.,a blood cell population of about 1 in 5 million or greater. In someembodiments, the technology can achieve a 35% capture rate and 90%, 99%or 99.9% purity from a sample with a mixed cell population, e.g., ablood cell population of about 1 in 50 million or greater. In someembodiments, the technology can achieve can achieve a 35% capture rateand 90%, 99% or 99.9% purity from a sample with a mixed cell population,e.g., a blood cell population of about 1 in 50 million or greater. Insome embodiments, the technology can achieve can achieve a 35% capturerate and 25%, 90% or 98% purity from a sample with a mixed cellpopulation, e.g., a blood cell population of about 1 in 500 million orgreater.

In some embodiments, the technology can achieve a combination of capturerate and purity above what is achieved with current methods. In someembodiments, the technology can achieve a 75% capture rate and 99.9%,99.99% or 100% purity from a sample with a mixed cell population, e.g.,a blood cell population of about 1 in 5 million or greater. In someembodiments, the technology can achieve a 75% capture rate and 90%, 99%or 99.9% purity from a sample with a mixed cell population, e.g., ablood cell population of about 1 in 50 million or greater. In someembodiments, the technology can achieve can achieve a 75% capture rateand 90%, 99% or 99.9% purity from a sample with a mixed cell population,e.g., a blood cell population of about 1 in 50 million or greater. Insome embodiments, the technology can achieve can achieve a 75% capturerate and 25%, 90% or 98% purity from a sample with a mixed cellpopulation, e.g., a blood cell population of about 1 in 500 million orgreater.

In some embodiments, the technology can achieve a combination of capturerate and purity above what is achieved with current methods. In someembodiments, the technology can achieve a 90% capture rate and 99.9%,99.99% or 100% purity from a sample with a mixed cell population, e.g.,a blood cell population of about 1 in 5 million or greater. In someembodiments, the technology can achieve a 90% capture rate and 90%, 99%or 99.9% purity from a sample with a mixed cell population, e.g., ablood cell population of about 1 in 50 million or greater. In someembodiments, the technology can achieve can achieve a 90% capture rateand 90%, 99% or 99.9% purity from a sample with a mixed cell population,e.g., a blood cell population of about 1 in 50 million or greater. Insome embodiments, the technology can achieve can achieve a 90% capturerate and 25%, 90% or 98% purity from a sample with a mixed cellpopulation, e.g., a blood cell population of about 1 in 500 million orgreater.

As described elsewhere herein, one embodiment of the MagSweepercomprises a round-bottom neodymium magnetic rod covered with an thin (25μm) non-adherent plastic sheath. The plastic material reduces thenon-specific binding of contaminant cells and does not impair magneticbinding. An exemplified member was constructed as a rod about 6 mm indiameter with a magnetic flux density of about 0.7 Tesla at the rod end.The magnet with the non-adherent sleeve sweeps the entire well in acontinuous motion to maximize the capture efficiency ofmagnetically-labeled cells. That is, a relatively large volumecontaining the mixed cell population can be contained in a well, whichhas a relatively shallow depth compared to a relatively larger diameteror width. In one embodiment a sleeved rod is robotically driven in apreprogrammed pattern to sweep through the well containing the sample ina pattern of overlapping concentric circular loops that cover theessentially the entire well area without scraping the walls of the well.It has been found that the choice of the velocity of the magnetic memberrelative to the solution (the sweep velocity) during the continuousrelative motion is important for to maximize (i) cell captureefficiency; (ii) application of sufficient shear force to detachadsorbed non-magnetically-labeled cells; and (iii) prevention of damageto the rare target cells. The optimum sweep velocity can also depend onthe magnetic strength of the A sweep velocity of about 2 mm/sec has beenfound to provide efficient capture.

The relative movement or motion between the magnetic member and thesample solution is generally a continuous relative movement. It is notsufficient to have the motion supplied by placing the magnetic memberinto and out of the solution. The movement must generally be acontinuous motion creating a shear across the magnetic member for a timesufficient for the magnetic member to contact a significant portion ofthe sample solution. The continuous motion is generally a gentle motionin the range of non-turbulent flow. The continuous motion is sufficientwhere it provides for the selective capture of rare target cells ascompared to contaminant cells in the sample solution.

We have found that the continuous relative movement having a velocitybetween the magnetic member and the solution of about 0.5 mm/sec toabout 5 mm/sec provides high capture efficiency. In some embodiments, arange of velocities between 1 mm/sec and 4 mm/sec is used. In someembodiments, the velocity between the magnetic member and the solutionrange of about 0.5 mm/sec to about 5 mm/sec when the surface magneticfield is in the range of between 0.1 Tesla and 1 Tesla. In someembodiments, the velocity between the magnetic member and the solutionrange of about 0.5 mm/sec to about 5 mm/sec when the surface magneticfield is in the range of between 0.1 Tesla and 1 Tesla. In someembodiments, these levels of velocity and magnetic field are used for amagnetic rod between 2 mm and 10 mm in diameter, for example with aneodymium magnet.

We have found that continuous relative motion should produce a velocitybetween the magnetic member and the solution which generally should be avelocity such that the system is in the non-turbulent flow regime. Anon-turbulent flow regime is a regime in which the flow of the solutionin the region of interest flows without creating turbulence. In somecases this can be the laminar flow regime. It is well known in the fieldof chemical engineering the factors that can be controlled in order tomaintain a non-turbulent flow regime. The factors include solutionviscosity, rate of flow, and geometrical factors. While not bound bytheory, it is believed that by maintaining a flow rate, i.e., relativemotion between the magnetic member and the sample liquid, which providesa non-turbulent flow regime, sufficient shear is provides in order towash off contaminant cells, but the flow is not so violent as to damagethe rare target cells, resulting in intact and/or viable cells aftercapture and release.

The appropriate velocity between the magnetic member and the solutioncan also be expressed in terms of Reynolds number. The Reynolds numberis a well known parameter in chemical engineering. We have found that aReynolds number between 0.1 and 100 can provide high capture efficiencyof intact or viable cells. In some embodiments, a Reynolds number ofbetween 0.5 and 50 is used. In some embodiments, a Reynolds number ofbetween 1 and 20 is used. In some embodiments, the Reynolds number isbetween 0.5 and 50 and the magnetic field is in the range of between 0.1Tesla and 1 Tesla. In some embodiments, the Reynolds number is between 1and 20 and the magnetic field is in the range of between 0.1 Tesla and 1Tesla. In some embodiments, these levels of Reynolds number and magneticfield are used for a magnetic rod between 2 mm and 10 mm in diameter,for example with a neodymium magnet.

The continuous relative motion between the magnetic member and thesample solution is also useful for ensuring that a significant portionof the sample has access to the magnetic member, or a significantportion of the sample experiences the magnetic field from the magneticmember sufficient to capture the magnetic particles in the solution. Insome cases it is desirable that a significant portion of the sample, amajority of the sample, or substantially all of the sample has access tothe magnetic member.

For example, in 1 ml of blood there are typically about 5 billion totalcells and 10 million white blood cells. This sample of blood may containdifferent quantities of target cells as noted below and these cells areisolated with the devices and methods of the invention with thefollowing purity levels:

-   -   Circulating Rare cells (e.g., fetal cells or circulating tumor        cells). Typically 5 to 1,000 cells per ml of blood. Typical        purity ranges from 50%-99.9% upon isolation with device        depending on starting quantity.    -   Stem cells (e.g., cancer stem cells). Typically ˜100,000 cells        per ml of blood. Typical purity ranges from 98%-100% after        isolation with device.    -   Neutrophils (e.g., neonatal immune response). Typically million        to 5 million cells per ml of blood. Typical purity ranges from        99%-100% after isolation with device.

Sequential rounds of capture-wash-release-recapture can be used toreduce or eliminate background contaminant cells that are notspecifically labeled with magnetic particles. In some cases, a second orthird round will improve the purity by but will do so at some cost tothe capture efficiency. Subsequent rounds are used for situations wherethe ratio of the rare target cells to the contaminant cells is high, forexample greater than 1 to 50 million.

Cell trajectories for the devices and methods of the invention under theinfluence of magnetic force and the fluid drag force were modeled. Notto be bound by theory, these models indicate that cell capture occurswhen the accelerated labeled cell through the magnetic force overcomesthe sweeping velocity and the corresponding viscous drag force of fluidflowing around the magnet. To find the capture zone around the magnet,numerical models that simulated the magnetic field, fluid flow, andtrajectories of labeled cells as a function of the sweeping parameterswere developed. By calculating the magnetic field using a finite elementmodel of the magnetic rod, it was found the magnetic flux is appreciable(>0.1 T) only near the rounded tip of the magnet and drops off rapidlyaway from the magnet. This result emphasizes the importance of employingthin non-adherent sleeves in order to minimize the attenuation of themagnetic field from the magnetic member and, consequently, improve theoverall capture rate. In addition, it was found that the magnet with therounded tip has an increased (50%) surface magnetic flux densitycompared with the unmodified (blunt tip) magnet. To estimate themagnetic force applied to the beads, a variable magnetic susceptibilityfor the beads was used, based on the manufacturer's bulk magnetizationdata. The travel velocity of labeled cells was calculated by adding themagnetic force-driven velocity, which was determined by Stoke's draglaw, to the fluid velocity, assuming a 10 μm diameter cell labeled witha single 4.5 μm diameter magnetic bead. The resulting 3-D fluid motionhas a significant impact on the particle capture. As illustrated in FIG.9, there exists a capture plane beneath the magnet indicated by dottedlines between arrow 908. There was a gap in the annular capture zone,even though the magnet has completed one full orbit. This is aconsequence of the “stifling” of the fluid by the magnetic member (rod),which pushes particles ahead of the magnet. At the higher z-planes,where magnetic fields are weak, there is a dramatic reduction in capturearea. Here, particles mostly travel around the magnet and through thewake without being captured. The device is therefore optimally designedto cover at least one circuit through the entire well, and mayadvantageously be swept through the well multiple times.

For scale, the maximum plotted velocity vector of the magnetic velocityfield is 7.8 mm/sec (at r=2 mm, z=−2.5 mm). The true maximum occursadjacent to the magnet surface at the tip (˜12 mm/sec). (c) Lateraltrapping boundaries for an idealized fluid-porous magnet as a functionof magnet sweeping velocity. (d) xy-trapping boundary 1.5 mm below themagnet tip (z=−4.68 mm) for the idealized flow. (e) Fluid velocitywithin the well calculated for a magnet velocity of 2 mm/sec and orbitradius of 6 mm. The vectors indicate the instantaneous flow velocity inthe xy-plane near the top of the curved section of the magnet (z=−0.2mm) and the color gives the flow magnitude. The fluid velocity field wasused to calculate particle trajectories and capture times. (f) Celltrapping profiles in the xy-plane located beneath the magnet (z=−4.8mm). (g) Cell trapping profiles at the fluid surface (z=3.5 mm). (h)Superimposed capture boundaries for one full orbit period (18.85 sec).

In some embodiments of the invention, no sample processing of blood isrequired prior to MagSweeper use, which decreases operator hands-on timeand risk for perturbing the CTCs. The MagSweeper can process 9 mlblood/hr with 3-5 minutes total hands-on time, and capture more than 50%of CTCs as measured in spiking experiments without significant change tothe captured cell gene expression. Another advantage of MagSweeper isthe flexibility in the starting sample volume and the processscalability. As a result, the throughput of the device can be increasedby sweeping an array of sheathed magnetic rods through multiple samplesin parallel with a single motion-controlled system. The device hassuccessfully purified 1-1.5 million neutrophils/ml of whole blood (datanot shown), which suggests that the MagSweeper is capable of purifying awide dynamic range of cell counts.

In regions of high magnetic field gradient (e.g., near the rounded tipof the magnet), labeled cells are efficiently captured, resulting in alarge sweep area. As noted above, the main influence of the fluid motionin this region is the presence of a small gap of uncaptured particlesleft following a single orbit. A simple first alternative is theextension of the sweep to overlap a portion of the circumference (˜10%),thus capturing the remaining particles. The effect of fluid motion isstrong in regions of low magnetic-field gradient, leading to asignificant reduction in capture cross-section. This is addressed withsmall steps (1 mm) in orbit radius.

This device has the demonstrated ability to efficiently capture livingepithelial cells from a sample, such as a blood sample (e.g., peripheralblood) while removing all contaminating cells. This exemplified devicecomprises magnetized members covered with non-magnetic, non-adherent,e.g., plastic, sleeves that can be robotically controlled to sweep or beswept through a blood sample that has been pretreated with a label, suchas Epithelial Cell Adhesion Molecule (EpCAM) antibodies linked tomagnetic particles. An EpCAM antibody is commercially available fromAbcam. It recognizes a 40 kD transmembrane epithelial glycoprotein(EGP40), also identified as human epithelial specific antigen (ESA) orepithelial cellular adhesion molecule (EpCAM). Cells or other labeledstructures are gently captured in the living state but at much highercapture and purity rates than any currently available commercialtechnology.

In some embodiment the devices of the invention utilize a “sweeping”effect to provide a continuous relative motion which causes the sampleto flow gently past the sleeved magnet. The sweeping effect can beaccomplished in a number of ways. In some embodiments, the magneticmembers immersed in the sample are moved in a predetermined patternthrough the while the container holding the sample is held stationary.Alternatively, the device may be constructed to move the containersrelative to the magnets. In other embodiments, the magnetic member withthe non-adherent sleeve can be held stationary while various solutionsare passed by the magnetic member. The present method and device can usea continuous movement through a substantial portion of the containerholding the sample, which movement causes at least a majority of thesample volume, in some cases, substantially all of the sample volume, tocontact the sleeved magnetic member. In other words, the movementpattern is designed to trace a pattern that traverses all or nearly allof the container area. The movement may be defined in an x-y horizontalaxis representing length and width (or, r representing a radius) with az dimension representing vertical. Movement will be primarily in an x-yor r direction, with different depths optimally provided along a z axis.The container may be a simple well-like container for holding anunobstructed pool of sample. The present method and device does notrequire the use of containers which comprise flow channels or aparticular fluid flow path; the sample is merely present in an open poolwhere it can be acted upon when the sleeved magnetic rod is insertedinto the pool. Target cell attachment to a side of the container is tobe minimized. The present method also does not require (but may employ)a pre-selection step based on cell size; the sample need not bepre-treated but may simply be diluted in buffer for convenience (e.g.,to prevent clotting) and extracted as-is. The present methods also donot require a chemical processing step, as does other methods, in whichthe CTCs are fixed and permeabilized, so the present method may resultin viable, intact cells. The present device further employs acontrollable robotic movement device for reproducible, predetermined,continuous patterned movement of the magnetic members relative to thesample.

In one embodiment, the magnetic members are alternatively magnetizableand de-magnetizable by contact with an electric field a magnet, or thelike. In FIG. 1, the magnetic members 106 a are magnetized, for example,through contact with a magnet contained in assembly 106. This isaccomplished by making the members 106 a out of magnetizable material.Magnetization occurs not just in materials having permanent magneticmoments but also in any magnetizable material in a field which caninduce a magnetic moment in its constituent atoms. In the special caseof Neodymium Magnet M=χH A m⁻¹.

Where M is magnetization and H is magnetic field strength, given inamperes per meter (A m⁻¹).

Thus, certain materials may be magnetized and demagnetized. The presentmagnetic member possesses high field strength in order to capture asignificant fraction of magnetically labeled cells.

The term “high field strength” as used herein generally means a fieldstrength that is capable of attracting and capturing the magneticallyresponsive particles. In some cases the field strength comprises amagnetic flux density of at least 0.3 Tesla, preferably at least 0.5Tesla, generally up to about 1 to 1.2 Tesla. In some embodiments, themagnetic flux density is measured at the point on the magnetic memberoutside the non-adherent sleeve that is the point of highest magneticflux density. In the case of a rod, the magnetic flux density ismeasured at the tip of the rod. Magnetic field strength maybe measuredas supplied by the magnet manufacturer, or by a gauss meter. The fieldstrength required, the high field strength, is also measured inpractical terms, such as by pull strength. The pull force may be testedby placing the magnet between two 1″ thick flat ground plates of alloysteel. One plate is attached to a digital force gauge which records thetensile force on the magnet. The plates are pulled apart until themagnet disconnects from one of the plates. The peak value is recorded asthe “pull force”. The present high field strength magnets will have apull strength of at least about 30 pounds, preferably at least about 70pounds. Effective high field strengths may be obtained, for example fromneodymium magnets.

Cell Surface Markers

Many different cell surface markers can be targeted with specificligands, and the ligands may be made magnetically responsive by a numberof reagents. For further information, see, for example, Stem Cells, Vol.25 No. 3 Mar. 2007, pp. 646-654, which discloses that humanfirst-trimester fetal blood, liver, and bone marrow MSC but not adultMSC express the pluripotency stem cell markers CD44, CD96, Nanog, Rex-1,SSEA-3, SSEA-4, Tra-1-60, and Tra-1-81. This enables the isolation ofrare fetal blood cells from maternal blood cells by the use of ligands(e.g., antibodies) to these cell surface markers. Also, patient specificstem cells may be isolated by the present method. Fetal nucleated redblood cells may be isolated, although rare, from maternal blood cellsusing anti CD71. For further information on isolation of fetal nucleatedblood cells, see Pittenger et al. (1999) Science, 284: 143-147 [3462].

Described below are findings from isolated single CTCs that wererecognized using antibodies to human EpCAM (epithelial cell adhesionmolecule). There are many cloned antibodies to EpCAM (formally calledTACSTD1 and also known as ESA). One of many monoclonal antibodies toEpCAM is HEA 125. For a list of others, seewww(dot)abcam.com/index.html?t=6920&pt=1&c=612.

Epithelial cell isolation techniques exemplified here can be applied toother sarcomas, adenosarcomas or carcinomas (epithelial malignancy),including prostate, breast, colon, colorectal and lung cancers. (See forfurther information on applicable malignancies, Clinical CancerResearch, Vol. 5, 4158-4163, December 1999. A more detailed listing ofcarcinomas is set forth in Allard et al., “Tumor Cells Circulate in thePeripheral Blood of All Major Carcinomas but not in Healthy Subjects orPatients With Nonmalignant Diseases,” Clinical Cancer Research, Vol. 10,6897-6904, Oct. 15, 2004, cited for further information. Carcinoma cellsare known to express the human Epithelial Antigen (recognized by themonoclonal antibody HAE125). Other human carcinoma antigens (HCA) areknown, as described e.g., in U.S. Pat. No. 5,693,763 to Codington, etal., issued Dec. 2, 1997, entitled “Antibodies to human carcinomaantigen,” which is cited for further information.

As another example, fetal cells may be isolated from maternal cells in amixed blood sample using an antibody to CD 34 (See US 20080254460).Also, a variety of cell surface markers are known to which antibodies orother labeling agents may be or have been made. These markers includethe above-mentioned CD 34, as well as CCR7, CD38, CD43, CD48, CD90,CD105, CD117/c-kit, CD123, CD135/Flk2, CD144 (VE-cadherin), CD150,CD2338/ABCG2, c-Met, Nanog, Notch-1, SSEA-1, SSEA3, SSEA-4, Tra1-60(podocalyxin) and Tra1-81 (podocalyxin).

Another stem cell marker is described in Bhatie, “AC133 expression inhuman stem cells,” Nature, 15:1685-1688 (2001), which is cited forfurther information, which reports that AC133+ cells are present in theperipheral circulation of mobilized and non-mobilized adults, and in thebone marrow compartment. These cells may be isolated and culturedaccording to the methods described here. According to the presentmethods, magnetic beads may be prepared having one maker, then cellsisolated using this marker may be further separated with a differentmarker, so that the resultant population comprises only cells expressingboth markers.

Bacteria and viruses may also be isolated even though present in smallnumbers in a cell population. For example, HIV present in low copynumber in a blood sample may be detected and isolated for furtherculture and genetic study by use of a specific monoclonal antibody suchas the monoclonal antibodies described in U.S. Pat. No. 6,818,392.

Removal of Cells

The captured cells are released from the covered magnetized members intoa recovery solution, such as a capturing buffer by removing the magneticfield across the non-adherent sleeve. To ensure removal of allcontaminating cells, the device may go through multiple rounds ofcapture, and release. In addition, wash steps are generally employedbetween capture and release steps. The cells released from the sleevehave been shown to be viable and unaltered. The beads may be removedfrom the isolated cells through the use of a cleavable linker betweenthe beads and the bound antibody for example. Chemically cleavablemoieties that may be incorporated into the linker include:dialkoxysilane, β-cyano ether, amino carbamate, dithoacetal, disulfide,and the like. Also, nucleic acid linkers can be used, with restrictionenzyme or other endonuclease recognition sites built in to the sequence.For a further description of cleavable linkers, see U.S. Pat. No.6,664,079 to Ju, et al., issued Dec. 16, 2003, entitled “Massiveparallel method for decoding DNA and RNA.”

Magnet Arrangements

Preferred embodiments of the device comprise a collection of axiallymagnetized rods of larger diameter compared to the above-describedwires. These may be 1 to 10 mm, 2.5 to 7 mm in diameter, or 4-6 mm indiameter, and are significantly higher in magnetic field strength thanthe needle configuration. The rods can be attached to a 5 function/3axis robot for moving the rods in a predetermined motion in a samplewell that is of much greater cross sectional size than the magnetic rod.Upon activation of the robot, the magnetic rod is inserted into an thin,tightly fitting plastic sleeve and lowered into a liquid sample (e.g.,human blood) that has been previously mixed with magnetic beads attachedto antibodies specific for a target biomarker (e.g., cancer cellslabeled with immunomagnetic beads). The robotic arm then sweeps themagnetic rod through the liquid to capture the magnetically labeledtargets (e.g., cancer cells). The robotic arm lifts the magnetic rod andcaptured biologics and then moves in a linear direction to the nextstation, where it lowers into a washing solution. The rodagitates/sweeps within the wash solution, during which time contaminantsfall off the magnetic rod. The wash solution may be drained and refilledmultiple times to ensure complete removal of contaminants. The robotthen moves the magnetic rod to a final release station. Here themagnetic rod is lifted out of the plastic sleeve, removing the magneticfield across the non-adherent sleeve, leaving the plastic sleeve in therelease fluid. The robot may shake the release fluid and cells orbiologics on the plastic sleeve fall into the release fluid for furtheranalysis. Any of the capture, wash, or release steps may be repeated forcomplete capture and purity. In some embodiments, a second magnet isused during the release step to assist in releasing the beads from thenon-adherent sleeve.

Non-Adherent Sleeves

The surface area and the geometry of the magnet design and the type ofnon-adherent sleeve, such as a plastic sleeve, can be adjusted to reducenonspecific adherence of contaminating cells and captured cells to theplastic. The robot permits control over the number and stringency ofwashes to ensure complete clearance of contamination.

A variety of materials which are non-adherent to red or white bloodcells may be used.

U.S. Pat. No. 3,723,754, French patent publication 2,089,788 andJapanese application Ser. No. 45/75116, disclose a device for bloodstorage and for dynamic flow of blood there through. As described there,polyvinylidene fluoride has desirable properties which may be employedin the present device. A preferred material, PVC (polyvinyl chloride),may be flexible, i.e., mixed with plasticizers. Plasticizers such asDi-2-ethylhexyl phthalate (DEHP) may be used to form up to 40% of thedry weight of flexible plastic used. Generally, little or no plasticizeris needed because the non-adherent sleeve is thin and in some cases isself supporting.

Polyurea-polyurethane sleeves may also be used, and prepared as coatingsof U.S. Pat. No. 5,169,720 are prepared from high molecular weightisocyanate end-capped prepolymers substantially or exclusively comprisedof ethylene oxide units. At least 75%, preferably at least 90%, of theprepolymer units are oxyethylene-based diols or polyols having molecularweights of about 7000-30,000, with essentially all of the hydroxylgroups capped with polyisocyanate prior to formation of the hydratedpolymer coating.

In addition, ceramic materials have been described as compatible withblood. See, U.S. Pat. No. 6,158,984 describing ceramic materials.polyHEMA (hydrophilic hydrogel poly(2-hydroxyethyl methacrylate) is alsoknown to reduce cell adherence and may be used alone or in combinationwith other materials.

Non-adherent materials are also described in U.S. Pat. No. 6,663,584.One may therefore, according to the present teachings, adaptnon-adherent, physiologically inert, liquid-repellent polymer such asfluorocarbon, including the chlorofluorocarbon, polymers or siliconepolymers for use here.

In one aspect, it is important that the device employ a thin sleeve,which can still be fitted on to and removed from the magnetic member. Bythin it is generally contemplated that the sleeve will be on the orderof 1 to 1000 μm, preferably 10-300 μm thick, preferably about 10-30 μmthick. The thinness or thickness may be adjusted to control the degreeto which the magnetic field is transmitted through the sleeve and thestrength of the sleeve material. The sleeve will generally beself-supporting, in that the magnetic member will be fully applied(e.g., inserted), and at least partially removed (e.g., withdrawn) orwholly removed repeatedly during operation, requiring a certain degreeof structural integrity in the sleeve. It is not strictly required thatthe sleeve be rigid or immobile when the magnetic member is removed. Inorder to facilitate release of the beads, the sleeve should generallynot contain or retain a magnetic field, although it may be paramagnetic.

Where a sleeve is used as the non-adherent sleeve, the sleeve isgenerally designed to form a surface on the outside of the magneticmember which allows the magnetic field from the magnetic member toeither pass through the sleeve or which is temporarily magnetized itselfwhen and only when the magnetic member is in close proximity to thesleeve. It is preferred that the rods and sleeves will be cylindrical tofacilitate application and removal of the sleeves, but otherconfigurations, such as curvilinear, can be designed given the presentteachings.

Magnetically Responsive Particles

Various types of magnetic beads can be used for attachment to the cellsto be isolated, in addition to the exemplified CELLECTED Dynabeads®,which are available from Dynal. Dynebeads are Dynabeads® aresuperparamagnetic, monosized polymer particles. The kit contains 5 ml“CELLection”™ Dynabeads” (4.5 m) coated with the monoclonal antibodyagainst the human epithelial antigen EpCAM and DNase Releasing Buffer.

Streptavidin-coated paramagnetic beads (2.8 μm diameter, M-280) beadsmay be obtained from Dynal Corp. in Lake Success, N.Y.Streptavidin-coated colloidal ferrofluid magnetic particles, or “MACS”,beads may be obtained from Miltenyi Biotec Corp. in Auburn, Calif. Byusing streptavidin-coated beads, one may specifically attach these beadsto biotin-labeled antibodies or other cell type specific proteins. As anexample of this implementation, one may refer to the presently marketedBD IMag™ Cell Separation System. This system utilizes magnetic beadtechnology for enrichment or depletion of specific cell populations in aprepared sample. BD Biosciences Pharmingen provides antibody-labeledmagnetic particles for enrichment or depletion of leukocytesubpopulations. Similar particles may be prepared for stem cell markers.BD IMag particles range in size between 0.1 and 0.45 μm and are coatedwith BD Pharmingen monoclonal antibodies.

Magnetically responsive particles as used here may be magnetic,superparamagnetic or paramagnetic. Magnetically responsive particles arefurther described in U.S. Pat. No. 5,628,407 to Gilbert, et al., issuedMay 13, 1997, entitled “Method and apparatus for separation ofmagnetically responsive spheres.”

In one aspect, the present invention comprises the use of large magneticbeads. The term “large magnetic beads” means magnetic beads (>1 micronsto about 10 microns) in size. This is distinguishable from small(0.7-1.5 microns), or colloidal (<200 nm) magnetic particles, which arealso referred to as nanoparticles.

Cell Isolation and Study

The method described here also comprises a use of the present devicewhich permits the isolation of intact, viable cells, especially CTCs. Byway of comparison, an FDA-approved device was used with increasedstringency of the approved wash step to improve purity, but the capturedcells were “beat up” in the process and not suitable for furthermultiplex molecular analysis. Cell membranes were torn off the capturedcell with spillage of the cell's nuclear material that was intended foranalysis. The present device is used with specified sweeping conditionsand wash conditions that greatly improve the recovery of viable CTCs.

The present methods do not rely on detecting a specific nucleic acidsequence in a malignant cell in order to identify a cell as a CTC. Theyenable the isolation of an intact cell for study of multiple geneexpression. As described below, the present method may comprise furthersteps, once CTCs have been isolated in an essentially pure composition,of studying expression in one or more of those isolated cells ofstandard breast cancer biomarkers, such as ER (estrogen receptor,further defined at PNAS, Aug. 29, 2006 vol. 103 no. 35 13162-13167), PR(progesterone receptor, further defined in Molecular Endocrinology, 12(9): 1334-1342) and HER2 (human epidermal growth factor receptor 2, alsocalled HER2/neu or c-erb2, further defined in Proc. Natl. Acad. Sci.USA, 1999 Sep. 14; 96(19): 10869-10874). In work described below, it wasdetermined that ER positive primary tumors may contain ER negative CTCs.Also, a HER2 positive primary tumor may result in HER2 negative CTCs. Offurther interest, it is demonstrated below that the CTCs areheterogeneous—one CTC from patient B expressed HER2, while three othersdid not. It was also determined using the present cell separation methodthat metastatic breast cancer CTCs frequently express vimentin and CD44.Thus, the present method enables CTC study which may be used for furtherdiagnostic purposes, such as in metastatic breast cancer. If atherapeutic target is present or absent, such as EGFR, HER2, ER or PR,will determine whether or not such a targeted therapy is appropriate.Also, in that disease, one may use analysis of CTCs to classify them asrepresenting mesenchymal-like cancer stem cells (MSC) or partiallydifferentiated epithelial cells (PDEs), where the presence of lessdifferentiated CTCs represents a hallmark of progressive metastaticdisease.

Cancer-derived stem-like cancer cells obtained by the side populationtechnique are described in Patrawela et al., “Side Population IsEnriched in Tumorigenic, Stem-Like Cancer Cells, whereas ABCG2+ andABCG2− Cancer Cells Are Similarly Tumorigenic,” Cancer Research,65:6207-6219 (Jul. 15, 2005). Some of these cells were characterized asmesenchymal. Thus, using the present teachings, one may isolate CTCs andstudy those isolated cells further, which will lead to the isolation ofa putative mesenchymal-like cancer stem cell (MSC) which may beidentified as less differentiated mesenchymal cells, or according totraits identified in the literature as involving the epithelial tomesenchymal transition, or by gene expression studies as describedbelow. Further guidance on identifying MSCs is given in Mani et al. “TheEpithelial-Mesenchymal Transition Generates Cells with Properties ofStem Cells,” Cell, 133:704-715 (May 16, 2008). Further guidance onidentifying MSCs is given below under the heading “Isolation of CTCsfrom metastatic breast cancer patients,” where it is explained that thepresent methods include the detection of increased vimentin expressionin CTCs, where vimentin is a mesenchymal cell marker.

Another aspect of the present device and method involves multiple geneanalysis from a single cell, where a cell is isolated, and a number ofgenes (exemplified as 15-48 genes) are analyzed for expression level ina single cell. The analysis may involve steps where the cell's RNA ispre-amplified with a reverse transcriptase PCR method and then tested byamplification simultaneously in multiple chambers of a microfluidicdevice. Alternative target genes to those specifically exemplified hereare contemplated. For example, pAkt/mTOR and p4E-BP1, may be analyzed inCTCs obtained by the present method, see Akcakanat et al., “Comparisonof Akt/mTOR signaling in primary breast tumors and matched distantmetastases,” Cancer, 112:2352-2358 (2008).

An exemplary panel of genes suitable for CTC characterization ispresented below, where the full name, taxonomy and sequence may beobtained from NCBI under the GeneID given. GeneID. The Entrez GeneGeneID is unique across all taxa. One can therefore convert any GeneIDinto its current names by using the definitions provided in the fileavailable as ftp: ftp.ncbi.nlm.nih.gov/gene/DATA/gene_info.gz.

TABLE 1 PTEN IGF-1R HER2 (ERBB2) EGFR c-MYC PI3K Gene ID Gene ID Gene ID30300 Gene ID 1956 Gene ID (Phosphoinositide- 5728 3480 4609 3- kinase)Gene ID5294 AKT mTOR MAPK ER PR GSTP1 Gene ID 207 (FRAP1) (MAPKSP1) GeneID 2099 Gene ID Gene ID (this is AKT1 Gene ID Gene ID 8649 5241 29502475 BCAR1 ALDH1 CD44 Vimentin Notch 1 Galectin 1 Gene ID Gene ID 216Gene ID 960 Gene ID7431 Gene ID Gene ID 9564 4851 3956 HSP70 Glut 1 LOXVEGF VEGF-R BCL2 Gene ID Gene ID Gene ID 4015 Gene ID7422 (FLT1) GeneID596 3308 6513 Gene ID 2321 BAX Caspase 4 Cyclin B2 P63 Osteopontin MDR1 Gene ID 581 Gene ID837 Gene ID 9133 Gene ID 8626 Gene ID Gene ID 66965243 beta-ACTIN GAPDH RPLPO GUS TFRC G6PD Gene ID 60 Gene ID Gene ID6175 Gene ID- Gene ID Gene ID 2597 none-beta 7037 2539 glucuronidasereported gene Additional Genes Used in Figs Ep-CAM CK19 CRYAB FOXA1 VIMFoxC1 Gene ID Gene ID Gene ID 1410 Gene ID 3169 Gene ID Gene ID 40723880 7431 2296 CD24 Gene ID 100133941

The profile set should include standard markers, such as, for breastcancer, ER, PR, HER2, and a proliferation marker such as cyclin B2. Inaddition, markers can be chosen from biologically relevant processessuch as signal transduction pathways involved in tyrosine kinasereceptor activity, markers of hypoxia, markers of cancer stem cells,genes involved in oxidative stress, drug resistance, apoptosis, andepithelial-mesenchymal transition. Although a housekeeping/referencegene is not supposed to vary in different cell samples, many do. Forexample, GAPDH has been recently shown to vary when cells shift to apentose phosphate pathway during hypoxia, and G6PD levels can vary indifferent tissues. As an initial product, one should choose thehousekeeping genes listed in the bottom row of the table above, as fiveof these are used as reference genes in the FDA-approved 21-geneOncotype DX RT-PCR tissue assay from Genomic Health.

The methods exemplified below use the MagSweeper technology to captureand purify CTCs for multiplex expression analysis at the single celllevel, as well as determining single cell CTC heterogeneity in somepatients which may reflect heterogeneity among multiple metastaticsites. To perform single cell analysis, the exemplified method manuallyidentifies and extracts each individual CTC in a post-processing stepfollowing CTC capture and purification.

II. Magnetic Coupling and Wire Embodiment (“MagBrush”)

In this embodiment, the rods or magnetic members are not themselvesmagnetic, but are made magnetic through coupling to a strong magnet, or,alternatively, made with an electromagnet.

Referring now to FIG. 1A-D, a container 102 holds a sample fluid 103,such as blood, which contains a small population of cells 104 (e.g.,CTLs) containing a magnetically responsive label. A magnetic support 106comprises a magnetic or electromagnetic portion which imparts a magneticfield to a number of magnetizable wires 106 a which are attached to thesupport and inserted into a sleeve array containing a matching number ofsleeves 108 held on a sleeve support 107. Thus the wires 106 a, eachcovered by an individual sleeve 108 surrounding the distal portion ofthe wire is inserted in to the sample fluid 103. This motion iscontrolled by an automatable controller 110, which moves supports 106and 107 either together or separately, depending on the process stepinvolved. As shown in FIG. 1A, the controller 110 inserts the sleevecovered wires into the sample fluid 103 and then carries out apredetermined robotized sweeping in order to contact the cells 104 andallow them to gently become attached to the sleeves 108. Then, as shownin FIG. 1B, the cells 104, bound to the sleeves 108, are removed fromthe sample fluid. Next, as shown in FIG. 1C, the cells bound to thesleeves are inserted into a wash or recovery fluid 114, and as shown inFigure D, the wires 106 a are retracted from the sleeves 108 by anupward movement relative thereto, controlled by robotic controller 110.

The device of FIG. 1 (termed the “Magbrush”) was constructed from wiresattached to a permanent strong magnet. The sleeves were 0.5 mm thickpolypropylene plastic. Cells were labeled with EpCAM antibodies andmagnetic nanoparticles from Stem Cell Technologies using the EasySepHuman EpCam Positive Selection Kit following manufacturer's directions.The breast cancer cell line MCF7 and the promyelocytic leukemic cellline HL60 used in the experiments were purchased from ATCC.EpCAM/nanoparticle labeled MCF7 cells and HL60 cells were mixed invarious numbers and then used the MagBrush to capture the epithelialcells. We mixed several thousand HL60 cells with either 1200, 100 or 50MCF7 cells.

For testing, the known tumor cell line MCF7 cells, with labeled EpCAMbeads, were used first. A well of a 96 well plate was filled with 250 μlof buffered saline and a known number of MCF7 breast cancer cellslabeled with the magnetic nano-particles were added to the well. Asingle wire prototype MagBrush without plastic sleeves (FIG. 2A, showinga detail of the wire) was then inserted into the well and allowed toincubate for 15 minutes before it was removed (FIG. 2B, showing cellsattached to the wire). For a high starting number of cells (1,200) 93%of the cells were cleared, for the medium cell number (100) 96% of thecells were cleared, and when starting with 50 cells, all of the cellsfrom the well were cleared.

Because the wire was permanently magnetized, it was not found possibleto release the cells from this experiment. The MagBrush was fitted withthe PVC sleeves and capture and release experiments were repeated.Capture of the cells was done with the same efficiency as in the aboveexperiment (FIG. 2C, showing a wire in a sleeve), but by removing thewire from the sleeve, the cells would fall off the plastic sleeve withease (FIG. 2D, showing the sleeve without cells).

Finally, cellular isolation was done with sleeve coated wires with amixture of MCF7 and HL60 cells. The HL60 cells do not express the EpCAMsurface antigen. It was again possible to isolate similar percentages ofthe MCF7 cells, but also, inadvertently, some of the HL60 cells werecaptured. To rectify this contamination, multiple rounds of capture andrelease were used and allowed for a pure isolation of the MCF7 cells.After capturing the MCF7 cells with the MagBrush, the cells werereleased into a tissue culture dish with growth media and allowed togrow for several days. The cells were able to attach and grow, showingthat viability was not affected by the labeling, capture and releaseprotocol.

Thus it can be seen that the MagBrush isolates purified and livingepithelial cells with high efficiency from a mixture of epithelial andnon-epithelial cells. This device has the ability to purify living CTCsfrom whole blood and should facilitate subsequent biological analysis.In addition, this high-throughput assay should result in a faster andless costly isolation of CTCs than current methods.

III. Permanent Magnetic Rod Embodiment (“MagSweeper”)

This embodiment uses a powerful permanent magnetic rod illustrated inFIG. 3, as well as in FIGS. 7-9. The rod has a substantial diameter, asopposed to a wire, and is magnetized longitudinally to form a dipolewith one end where the beads are shown as attaching to the bottom of therod. The magnet has a very high pull force (e.g., ˜0.99 lbs) and may beon the order of ⅛ inch in diameter. The preferred magnetic rod is a rareearth magnet. Neodymium magnets are a member of the Rare Earth magnetfamily and are the most powerful permanent magnets in the world. Theyare also referred to as NdFeB magnets, or NIB, because they are composedmainly of Neodymium (Nd), Iron (Fe) and Boron (B). The magnets used inthe exemplified device were obtained from K&J Magnets, as model D2X0,with the following specifications: Dimensions: ⅛″ dia.×1″ thick;Tolerances: ±0.001″×±0.002″; Material: NdFeB, Grade N42;Plating/Coating: Ni—Cu—Ni (Nickel); Magnetization Direction: Axial(Poles on Flat Ends); Weight: 0.0532 oz. (1.51 g); Pull Force: 0.99 lbs;Surface Field: 4175 Gauss; Brmax: 13,200 Gauss; BHmax: 42 MGOe.

Referring now to FIG. 3A-F, there is shown a device operating inprinciple as the device in FIG. 1. The illustrated device is made up ofthree NdFeB magnetic rods. The rods are covered with a removable plasticsleeve that is designed to fit tightly over the rods (FIG. 3A). For cellcapture, the rods are engaged into the plastic sleeves and then movedthrough a blood sample treated with Dynabead® superparamagnetic,monosized polymer particles (FIG. 3B). The rod/sleeve assembly is thenrinsed in PBS several times then moved (FIG. 3C) into a fresh well wherethe cell containing rods are immersed (FIG. 3D). The cell-bearing rodsare moved to another container for receiving the cells (FIG. 3E), thenmagnets are removed from the sleeves and a magnet is placed under thesleeves to facilitate removal of labeled cells from the now nonmagneticsleeves (FIG. 3F). Attachment and removal of labeled cells can be seenat the bottom of FIG. 3F.

Three containers 304 each receive a single magnetic member. The threemagnets are constructed in a magnet array 308, where each magnetic rodcan be removably enclosed within a sleeve 306 which is between themagnetic member and the sample and contacts the sample. As shown in FIG.3B, the sleeved magnets are swirled through the sample, and magneticallyresponsive cells adhere to the magnets (through the sleeves), as shownat 312. Although the cells are shown as being concentrated near thedistal tip of the rod, the cell attachment will vary depending on thedesign of the magnet (See FIG. 8). Once the cells have attached to theplastic sleeve, the assembly is removed from the container containingthe cells to be captured, as shown at FIG. 3C. As shown at FIG. 3D, theassembly is next moved into a wash container having a buffer 317 forremoving any contaminating cells which may have nonspecifically bound tothe sleeve. This step may be repeated as needed. Then, as shown in FIG.3E, the assembly is moved to another solution 318 into which the cellsmay be released and captured. As shown in FIG. 3F, the magnets 306 areremoved from the sleeves 306, and release of the cells is assisted bymagnets 320 on the bottom of the wells, causing the cells bearingmagnetically responsive labels to be drawn to an area 322 adjacent themagnets 320.

In order to obtain a magnet design that has extremely high captureability in the design utilized, a series of experiments were conducted,as illustrated in FIG. 8. Different magnet configurations are shown atFIG. 8A through FIG. 8F, showing a single rod in each figure withvariations in axial segmentation and diameters below the magnet support.In order to test different magnet configurations, a mixture was preparedwith ˜10,000 MCF7 cells in 2 ml of DMEM which was treated with 80 μl ofEpCAM for 15 minutes, 80 μl of nanoparticles for 15 minutes, and 15minutes in robosep magnet. Supernatant was removed and labeled cellswere resuspended in 1 ml DMEM. Count (7.5 cells/μl). 10 μl of cells wasadded to 2.5 ml of DMEM into each of 12 wells of a 24 well plate. Thecells were swept with the robot then the procedure was to spin downplate at 800 rpm for 5 minutes and then count cells in the wells. Thenthe designs in FIG. 8 A-F were tested as follows:

a) Two wells got no magnets—51, 32

b) Two wells got nonmagnetized 5.1 mm cylinder—13, 7

c) Two wells got 3 mm (⅛ inch) cylinders—1, 2

d) Two wells got 1.5 mm ( 1/16 inch) cylinders—9, 22

e) Two wells got 3/16″ ring magnets wrapped in parafilm—0, 0

f) Two wells got 3/16″ ring magnets—1, 0.

The use of a single long magnet, with a longitudinal dipole (FIG. 8D),was found here to give superior results.

In order to demonstrate the ability of the present device to separatetumor cells from blood, a known number of MCF7 cells stably transfectedwith pAcGFP1-N1 from Clontech were added to 9 ml of normal donor blood(GFP labeling was done for easier epithelial cell identification). 10 mlof CELLection Epithelial Enrich Dynabeads were added and incubated for30 minutes at room temp with constant mixing. The device was thenutilized to isolate and purify the cells. Blood samples were collectedfrom female patients with metastatic breast cancer in compliance withall regulations regarding Human Subjects. 9 ml of blood from eachpatient was collected, labeled with Dynabeads, isolated with the Deviceas above. Single CTCs were manually selected for multiplex real-timeqRT-PCR. For qRT-PCR, the single cells were pre-amplified for 15 targetswith qRT-PCR kit from Invitrogen. Each of the pre-amplified products wasdiluted and applied to qRT-PCR reactions for each individual targetlisted below. We used TaqMan primers and probes from Applied Biosystems.Human reference RNA and TE buffer were used as controls.

Referring now to FIGS. 7A and B, which is a drawing corresponding to anas constructed prototype, there is illustrated a support plate 106 whichis attached to and holds three magnetic rods 106 a, which extenddownwardly through a sleeve array, and a sleeve holder which maintainsthe sleeves in a fixed position relative to withdrawal and insertion ofthe magnets. The axially or diametrically magnetized rods are attachedto a 5-function/3 axis robot. The top view shows the approximatedimensions of the array in a six-rod prototype embodiment. Uponactivation of the robot, the magnetic rod is inserted into a plasticsleeve and lowered into a liquid sample (e.g., human blood) that hasbeen previously mixed with magnetic beads attached to antibodiesspecific for a target biomarker (e.g., cancer cells labeled withimmunomagnetic beads). The robotic arm then sweeps the magnetic rodthrough the liquid to capture the magnetically labeled targets (e.g.,cancer cells). The robotic arm lifts the magnetic rod and capturedbiologics and then moves in a linear direction to the next station,where it lowers into a washing solution. The rod agitates/sweeps withinthe wash solution, during which time contaminants fall off the magneticrod. The wash solution may be drained and refilled multiple times toensure complete removal of contaminants. The robot then moves themagnetic rod to a final release station. Here the magnetic rod is in,but can be lifted out of the plastic sleeve, which still remains in therelease fluid. The robot shakes the release fluid and cells or biologicson the plastic sleeve fall into the release fluid for further analysis.Any of the capture, wash, release steps may be repeated for completecapture and purity.

IV. Fluid Flow Embodiment—Stationary Non-Adherent Sleeve

An alternative embodiment utilizes fluid flow, for example in a fluidicchannel. For example, the non-adherent sleeves comprising the magneticmembers extend into a fluidic channel. The various sample, wash, andrelease solutions are then flow past the non-adherent sleeves. Themagnetic field across the non-adherent sleeves can be applied andremoved in order to capture particles from, or release particles intothe fluid.

As with the other embodiments described herein, controlling the velocitybetween the magnetic member and non-adhesive sleeve during capture isimportant in order to obtain the purities and capture efficienciesdescribed herein. The same ranged of velocity and Reynolds numberdescribed above are relevant to this embodiment as well. As above, it isdesired to have fluid flow rates around the magnetic member that are inthe non-turbulent flow regime.

FIG. 15 shows an example of this embodiment. Here, magnetic members 1502extend into a fluidic channel 1506 containing a liquid sample of cells1508 within the channel, which can flow in a direction indicated byarrow 1510. The magnetic members 1502 are surrounded by a non-adherentsleeve 1504, which can be formed within the fluidic channel 1506, wherethe magnetic members would contact the solution 1508 due to theirimpingement into the channel, but for the sleeve 1504. The magneticfield from the magnetic member across the non-adherent sleeve can beapplied and removed for capture and release. In some embodiments, themagnetic members are stationary throughout the process, and the magneticmember is magnetized and de-magnetized, for example using one or moreelectromagnets. In other embodiments, the magnetic member is insertedinto the non-adherent sleeve to apply the magnetic field, then pulledout at least partially so as to substantially remove the field acrossthe non-adherent sleeve. The embodiment in FIG. 15 shows 3 magneticmembers, but any effective number of magnetic members can be used. Insome cases one magnetic member can be used, in other cases numbers up to10, 100 or more can be used.

In some embodiments, the apparatus comprises a fluidic channelcontaining a hollow pillar or an array of hollow pillars, in which anexternal magnet engages or withdraws in each pillar (see FIG. 16(A)through (D)). By engaging magnets inside hollow pillars, magneticallylabeled cells 1602 will be attracted and attached to pillars resultingin capture (FIG. 16B). By withdrawing the magnets from the pillars, thelabeled cells will detach from the pillars and get released to the bulksolution in the channel (FIG. 16D). In some cases a wash step can alsobe used by passing wash solution while the magnetic member is engaged(FIG. 16C). To facilitate the target cell removal from the pillars, anexternal magnet can be placed under the fluidic channel and/or thefluidic flow can be adjusted (FIG. 17). In some embodiments, eachchannel can contain one or an array of hollow pillars (FIG. 16).Magnetized pillars can go freely travel inside and outside these pillarsfor capturing and releasing the targeted cells labeled withfunctionalized magnetic beads. When the magnetized pillar enters thehollow pillar in the fluidic channel, the labeled cells will attach tothe pillar surface (FIG. 16B). After washing the fluidic channel with awash buffer (FIG. 16C), the magnets will disengage from the hollowpillars to release the captured cells (FIG. 16D). In some cases, thereleased cells go through rounds of capture-wash-release to increase thepurity of target cells. The fluid flows in the direction of arrow 1604,and it can be seen that the released cells flow downstream. Theembodiments of FIG. 16-18 further contemplate additional fluidcompartments to deliver and receive the fluid. The fluid may further becontinuously circulated or washed back in forth in a reciprocal manner.This provides multiple sample contact and wash steps. The sample fluidmay be contained in compressible chambers, for example, at either end ofthe illustrated tube for receiving and expelling fluid under controle.g. of mechanical plungers or rollers, which operate in a continuousmanner or according to other preprogrammed sequences.

FIG. 17 shows the magnetic member 1702 in a fluid channel 1704 similarto similar to FIGS. 15 and 16. To assist the release of target cells,the flow rate of the fluid can be increased (FIG. 17B) or an externalmagnet 1706 can be placed underneath the fluidic channel (FIG. 17C) andunderneath the magnetic member. This external magnet can be a permanentmagnet which can be mechanically placed and removed from underneath thechannel or it can be an electromagnet which can be turned on and offelectrically.

In one alternative embodiment, shown in FIG. 18, the fluidic channel1802 comprises a thin and elastic wall. When the magnet 1806 is pushedtoward and into the fluidic channel, the fluidic wall will also deformand form a hollow pillar around the magnet, contacting the solution1804.

V. Procedures

General Procedures and Robot Control

As described in detail below, the present methods involve firstobtaining a sample containing cells or other structures to be isolated(target cells), such as the isolation of tumor cells (which are rare,i.e., 1 in 100-10,000 blood cells in peripheral blood). The blood sampleis exposed to a reagent which labels only the target cells. Theantibodies are attached to a magnetically responsive particle, such asby biotinylating the antibody and binding it to a streptavidin-coatedbead. A preferred reagent is an antibody that recognizes tumor cells.The tumor cells will be of a different phenotype than the blood cells,such as epithelial cells, as are a typical breast cancer cell type.Other tumors may be of endothelial or connective origin and may bedistinguished on that basis. Other labels may be based on tumor or cellsubtype, such as markers for stem cells, or certain tumor phenotypes,such as EGF+ or Her2+, etc. The labels are attached to a magneticparticle and thoroughly mixed in the blood sample. Then the magneticmembers, encased in inert nonmagnetic sleeves which permit the magneticforce to pass through, and, further, do not absorb or adhere to theblood cells or target cells, are contacted with the blood sample. Thesleeved members are thoroughly exposed to the blood sample so as to notdestroy cells, but come into close contact with any rare cells present.

In some cases, when running the capture protocol, the magnets areapproximately 1-2 mm from the bottom of the plate in the containerholding the blood sample. The sleeves coating the magnetic members aremade, for example, of PVC are fitted very tightly to the magnets. Thethickness of plastic sleeves are in the range of 0.001-0.01″ (25micron-254 micron). During the capture phase (see FIGS. 1A and 3B), arobotic arm attached to an actuator capable of movement in threedirections (xyz actuator) sweeps through each sample container in apredefined, reproducible pattern. A robotic arm moves at 2 mm/second, itmakes 12 loops, and each loop has a radius offset of 1 mm from the priorloop. Next, as shown in FIG. 1B and FIGS. 3C and 3D, the magneticmembers are moved into a different solution for washing. This ispreferably accomplished by moving the robot, but the sample holder mayalso be moved. The arms are immersed in a completely different liquid towash off non-target cells that have become entrained in thebead-decorated cells or non-target cells that have non-specificallybound to the inert sleeve that covers the magnets. For washing, therobotic arm preferably moves, e.g., at 2 mm/second, over a loop radiusof 5 mm and goes for 30 seconds. Washing is preferably carried outseveral times by changing the solution in the second container. Next,after washing, the target cells, with magnetic beads attached, arereleased into a third container as a purified population. This is shownin FIGS. 1C and 1D and FIGS. 3E and 3F. The cells are typically placedinto a capture buffer in a third container, which has magnets at thebottom to help draw the magnetic beads off of the inert sleeves. At thisstage, the magnet is removed from the sleeve so that the beads are nolonger drawn to the sleeves and will fall off. At release, there is noarm movement. The sleeve covered magnet enters the liquid, the magnetsare withdrawn and there is a strong magnet (the exemplified magnet is aneodymium rod magnet, e.g., as made by K&J Magnetics, Jamison, Pa.)underneath the plate that pulls the cells/beads off the sleeves to thebottom of the plate.

As can be seen from the above description, the entire process can beautomated by computer control, once the blood has been labeled byantibodies specific to the cells to be removed, preferably CTLs.

Exemplary Procedure for Obtaining Rare Cells from Human Blood

As an exemplary protocol for obtaining rare cells from human wholeblood, blood was collected in 10 ml EDTA vials (BD#366643). About 9 mlper each vial was actually obtained. Blood is then taken to the lab andthe blood is split into three (3) new vials and each is brought up to 6ml total with PBS pH7.2. Next 3.4 ul of Dynabeads, Cellection EpithelialEnrich (4.5 um diameter superparamagnetic polystyrene beads coated withmonoclonal mouse IgG1 antibody Ber-EP4 [aka EpCAM]) are added to eachsample. The samples are then mixed for 30 minutes at room temperature,while being rotated (10 rotations/minute). Samples are then placed intowells of a 6-well plate (Falcon 353502) and the tubes are rinsed withPBS to bring each sample up to 10 ml. The robot is then run as follows:

Two capture cycles, two wash cycles, one release cycle. The platecontaining the released cells in then placed in the capture position andone round of capture is run. This recapture is how we reduce/avoidcontamination by blood cells.

The samples are then washed twice (that is repeating steps shown inFIGS. 3C and 3D a multiple number of times) and then released into 500ul tubes containing 360 ul of media (300 ul DMEM with 10% fetal bovineserum, 30 ul of DNase1, 30 ul of 25 mM magnesium chloride). This removessome of the beads, but mostly it just keeps beads from stickingtogether, which minimizes clumps so that the cells are easier to see.

Samples are then incubated for 10 minutes at room temperature and thenthe samples are transferred to a 6-well plate, where cells are handselected (picked up using a micropipet). Each cell is collected in avolume of 2 ul and with 0.2 ul of RNase inhibitor (Superase IN, fromAmbion, product #2694) added. Samples a then frozen on dry ice until theqRT-PCR is run.

TABLE 2 Examples of MCF7 cells spiked into normal donor blood, labeledwith Dynabeads and captured using the Device. Cells Added Cells Isolated41 8 50 10 40 8 24 13 16 7 19 5Isolation of Breast Cancer Tumor Cells from Patient Blood and Analysisof Those Cells by rt-PCR

Two patient samples are described. From the first patient (ID# SM014),one vial of blood was obtained. From this patient, 10 potential cells,were isolated, but only one of which had RNA of good enough quality toget data from the qRT-PCR. For the second patient, (ID#SUBL017), threevials of blood were received, and isolated 26 potential cells wereisolated, of which 25 showed RNA.

Cells have since been isolated from 4 additional patients (82 potentialcells) and from the blood of mice that were growing tumors derived froma human breast cancer cell line (MDA-MD-231). From the mice, 16 cellsamples were subjected to qRT-PCR analysis and several hundred morecells were grown in culture.

Pre-Amplification for Multiple Targets

1. Samples were pre-amplified with the CellsDirect™ q RT-PCR kit(Invitrogen, catalog Number 11754-100). For Multiplex qRT-PCR, eachassay employed a TaqMan assay from Applied Biosystems. The 20× assaysfor the targets were diluted in TE buffer to make 1× Assay Mix and usedin the pre-amplification step.

The sample RT-Pre-Amp Master Mix was prepared by combining the followingsteps and components:

Component Volume* (μL) CellsDirect 2X Reaction Mix 5.0 lx Assay Mix 0.5RT/Taq Enzyme 1 Cells in TE buffer 3.5 Total 10 Note: If the cells areto be stored after sorting or, if the RNase activity is suspect, add 0.1μL of Ambion's SUPERase-In

2. Perform Thermocycling.

-   -   a. Reverses transcribe the RNA to cDNA at 50° C. for 15 minutes.    -   b. Inactivate the RT enzyme and start the Taq by bringing the        sample to 95° C. for 2 minutes.    -   c. Preamplify the cDNA by denaturing for 18 cycles at 95° C. for        15 seconds each, and annealing at 60° C. for 4 minutes.    -   d. Dilute the resulting cDNA product 1:2 with water or TE        buffer.        Application of the Preamplified Sample on Fluidigm Biomark™        48.48 Dynamic Array Chip

This was done for multiple qRT-PCR reactions based on the manufactureinstructions. This chip is commercially available and is describedfurther on the Fluidigm web site. It carries out TaqMan® PCR assays in amatrix of channels and valves on a microfluidic chip. 48 samples and 48assays can be loaded into the inlets of the chip's input frame. Thispermitted analysis of single cells, which contain picogram quantities ofRNA, insufficient for reproducible microarray analysis. The chip usesintegrated fluidic circuits and pressure-controlled nanovalves toperform highly sensitive parallel qRT-PCR assays based on standard5′-nuclease probe (TaqMan) chemistry and primer-probe design rules. Therows of these chips are loaded with the reagents (primer pairs plusTaqMan® probe mixtures, for instance) and the columns with cDNA samplesgenerated from cellular RNA and other PCR components that are preparedoff chip. The contents of the rows and columns are then mixed with eachother automatically in the designed reaction chambers of the chip (10 nleach) without cross-contamination. Therefore the primers and samples canbe tested in a combinatoric manner: systematically combined into 2,304parallel reactions. Performing the same set of reactions by hand or witha robot would require orders of magnitude more reagents and pipettingsteps, each of which could introduce the possibility of mistake orcross-contamination. Moreover, the quantity of costly reagents used ismuch lower than with standard qRT-PCR.

Single cell analysis may be conducted in a number of alternative ways.For example, one could carry out a smaller set of PCR or otheramplification reactions; one could culture the single cell to obtain alarger RNA mass; and one could test the single cell with enzymatic orimmunological reagents.

Results of qRT-PCR from Tumor Cells Isolated According to the PresentMethods

The results, obtained from patient samples, and using qRT-PCR describedabove, are shown in Table 3 below, and in FIG. 6A-E. Table 3 showsrelative expression (in percentage) of 15 genes from multiplex real-timeqRT-PCR experiments comparing 0.1 ng of human reference RNA (set to 100%expression) to a single MCF7 cultured cell, and a representative sampleof isolated circulating tumor cells (CTCs) captured from the blood oftwo cancer patients (25 single CTCs were individually analyzed in thesecond patient). Both patients have high-grade infiltrating ductalcarcinomas with metastases.

The primary tumor of patient SM014 was ER−/PR−/HER2+. Bone metastasesprogressed on trastuzumab, and a biopsied bone metastasis wasER−/PR−/HER2−, which matches the expression profile of the CTC SM014cell 7. Patient SUBL017 had a primary left breast cancer that wasER−/PR+/HER2−, then several years later developed bilateralsupraclavicular and mediastinal adenopathy. A right SCLN biopsy wasER+/PR−/HER2−. Two CTCs from this patient, who now has bone metastases,are ER−/PR−/HER2−. All cells were positive for CK19, but showed a widerange of expression levels for the other genes tested.

Measurement of Expression Profiles from CTCs Extracted by a MagSweeperfrom Eleven Women with Metastatic Breast Cancer

TABLE 3 0.1 ng RNA MCF7 single cell SM014-7 SUBL017-18 SUBL017-23 Target% % % % % GAPDH 100 3.28 0.06 0.06 0.17 Beta Actin 100 21.92 N/A N/A0.24 Big ribosome protein (RPLPO) 100 21.76 0.29 N/A 0.9 CRYAB 100 N/AN/A N/A N/A EGFR 100 N/A N/A N/A N/A FOXA1 100 271.31 N/A N/A N/A CD44100 10.73 N/A N/A 0.61 ESR1 (ER) 100 606.29 N/A N/A N/A PGR (PR) 100207.05 N/A N/A N/A Vimentin (VIM) 100 N/A N/A 0.95 N/A ERBB2 (HER2) 10072.7 N/A N/A N/A EpCAM 100 14.97 3.35 N/A N/A FOXC1 100 8.42 N/A 8.36N/A CD24 100 26.79 N/A 2.11 N/A CK19 100 772.75 143.4 22.07 19.75 TEBuffer N/A N/A N/A N/A N/A N/A = not amplified.MagSweeper Parameters

The device used in this example has been described above. Moreparticularly, it utilized powerful neodymium magnetic rods with roundedbottoms. The rods were 6 mm in diameter (50 mm² capturing area) and amagnetic flux density of 0.5 Tesla at the rod end. The magnetic rods areattached to an actuator to sweep through a blood sample and extractimmunomagnetically-labeled tumor cells. During capture, the rods arecovered in a detachable, ultra-thin (˜25 micron) inert plastic composedof polyvinyl chloride (PVC). As described previously, with the magneticrods engaged inside the plastic sleeve, the labeled cells are attractedto the plastic sleeve; disengaging the rod from its sleeve removes thestrong magnetic field gradient and facilitates easy removal of labeledcells. PVC was selected for its lack of nonspecific blood cell bindingfrom among 21 sleeve materials tested.

Magnets used in the MagSweeper were purchased from K&J Magnetics, Inc.(Jamison, Pa.). The sleeved rods were robotically driven to sweepthrough wells containing blood samples (one rod per well) in a patternof overlapping concentric circular loops that covered the entire wellarea. The sleeved magnetic rods sweep through the sample in overlappingconcentric circles at 2 mm per second, capturing labeled cells and freemagnetic beads, as well as some contaminating blood cells. The sweepvelocity was selected for i) cell capture efficiency; ii) application ofsufficient shear force to detach adsorbed non-magnetically labeled cells(wash step FIG. 3D); and iii) prevention of damage to the fragile CTCs.It was found experimentally that an optimal circular velocity was around2 mm/second. After cell capture, the sleeved rods with attached cellsmove to a new well containing phosphate-buffered saline (PBS). The cellswere circularly swept through the wash buffer solution to removeunlabeled blood cells which may be adsorbed to the rods. Finally, therods and cells were moved to a third wash well containing fresh PBS. Thecells were released into the PBS by withdrawing the rods from thesleeves and applying an external magnetic field under the well.Magnetically-labeled cells easily migrate off the plastic sleeve towardthe bottom of the well, releasing unlabeled blood cells that may havebeen trapped between labeled cells and excess magnetic beads. Afterre-engaging the rods into their plastic sleeves, the labeled cells arethen re-captured with many fewer entrapped contaminating blood cells.Sequential rounds of capture-wash-release-recapture significantlyimprove purity and eliminate all blood cell contamination.

Samples loaded into 6-well plates were placed into the capture positionof the MagSweeper. The magnetic rods engaged inside their plasticsleeves were programmed to sweep through the immunomagnetically-labeledblood sample at 2 mm/sec in 13 concentric loops, each loop offset by 1mm. The capturing was immediately repeated. The sleeved magnetic rodsthen moved to a wash station where they entered and swept through 10 mlof PBS for 30 seconds and then washed again in 10 ml of fresh PBS. Therods then entered a fresh 6-well plate containing PBS at the releasestation. The rods were then disengaged from their sleeves. This releaseplate was then moved to the capturing position and thecapture-wash-release protocol was repeated at least one additional timeusing a fresh sleeve.

Procedure for Labeling and Single Cell Analysis

To capture CTCs, the CTC cell membrane was labeled with 4.5 μmparamagnetic beads (Dynabeads) functionalized with anti-EpCAMantibodies. Large magnetic beads permit isolation of cells with only onemagnetic bead attached, and reduce the need for high EpCAM antigenexpression by the CTCs. Cells may only have one bead attached, or mayhave multiple beads attached.

The MagSweeper platform allows visual verification of CTCs released intosolution; these CTCs, free from contaminating blood cells, wereindividually extracted by pipette aspiration, thereby permitting singlecell analysis. While another technology, cell sorting, can also producepure single cells, many cells are adsorbed in the tubing, thus requiringan input of hundreds of CTCs, making current sorting technologiesimpractical for single cell CTC analysis for most cancer patients.

CTCs were visually identified and photographed using an Axio Observer A1inverted microscope (Carl Zeiss MicroImaging, Göttingen, Germany).Single cells were manually aspirated using a Pipetman P2 (Gilson, Inc,Middleton, Wis.) under visual guidance. Cells were collected in 1 μlvolume and added to 0.2 μl of SUPERase-In™ RNAse inhibitor (AppliedBiosystems/Ambion, Austin, Tex.) and frozen on dry ice. Samples werestored at minus 80° C. until processed.

These microscopic observations demonstrated that the present method anddevice produced a pure composition of CTCs, with zero contaminatingblood cells such as white blood cells, red blood cells, platelets or thelike. In the present methods, using a clinically realistic human bloodsample of about 9 ml, a composition comprising at least 10 CTCs can beobtained, representing a new composition of essentially pure (at least90% pure, preferably 95% pure and often 100% pure) human CTCs obtained,as implied by the definition of CTCs, from human peripheral blood. Thiscomposition was seen as free of extrinsic cells by microscopicexamination.

The performance of the MagSweeper was tested by measuring captureefficiency and cell purity by spiking 5-50 magnetically-labeled cellsfrom MCF7 and SKBR3 human breast cancer cell lines into normal wholeblood and processing the samples with the MagSweeper. In replicateexperiments (n=9 for each cell line tested), it was found that the cellcapture rate was 59%±27% for MCF7 cells and 66%±17% for SKBR3 cells. Inall cases, cells were isolated with 100% purity after two rounds ofcapture-wash-release as determined by visual inspection (zerocontaminating WBCs).

Pilot Studies Showing Viability and Gene Expression of Captured Cells

To study the ability of the MagSweeper to capture CTCs in aphysiological setting, a model system was used to produce CTCs in mice.Orthotopic tumor xenografts in twenty immunocompromised (NOD-SCID)female mice were created by injecting their mammary fat pads withMDA-MB-231 human breast cancer cells. Fifty days post injection, theaverage tumor volume was 1.5 cm³ and macroscopic lung metastases wereobserved in all mice. Control mice injected with saline did not developprimary tumors or metastases. Mouse blood was labeled withimmunomagnetic beads (against human EpCAM antigen) and processed withthe MagSweeper. Greater than 100 CTCs were captured from the blood ofeach tumor-bearing mouse but no CTCs were captured from the blood ofcontrol mice. The CTCs isolated by the MagSweeper retained their abilityto grow in culture, again confirming CTC viability. Single cellbiomarker profiles of all captured CTCs matched single cells from theparental cell line used to generate the tumor xenografts. Similar to thein vitro experiments, these mouse experiments demonstrated that highquality CTCs could be captured in vivo without altering gene expression.Again, 100% purity of all extracted CTCs was visually confirmed. Theresults shown in FIG. 11 indicate the integrity of the RNA from theisolated CTCs.

Single Cell Analysis

To determine the effect of the present capture method on CTC geneexpression profiles, single captured cells, which generally containpicogram quantities of RNA, were analyzed with high-throughputmicrofluidic chips that use integrated fluidic circuits and integratedmicromechanical valves to perform highly sensitive parallel qRT-PCRassays (Fluidigm 48.48 Dynamic Array, described further in J. Liu, C.Hansen, Quake S. R. Anal. Chem. 75, 4718-4723 (2003) and S. L. Spurgeon,R. C. Jones, R. Ramakrishnan, PLoS ONE 3, e1662 (2008).)

Samples were pre-amplified with the CellsDirect qRT-PCR kit (Invitrogen,Carlsbad, Calif.). The 15 TagMan expression assays (20×) (AppliedBiosystems, Foster City, Calif.) were performed for the following genes,where the number in parentheses set forth sources of furtherinformation, in the form either of an Applied Biosystems catalog number(genes 1-3) or a gene symbol, Gene hCG2043341 Celera Annotation:

1. Human GAPD (GAPDH) Endogenous Control (4333764F);

2. Human ACTB (beta actin) Endogenous Control (4333762F);

3. Human RPLPO (large ribosome protein) Endogenous Control (4333761F);

4. ESR1 (ER, Hs00174860_m1);

5. PGR (PR, Hs00172183_m1);

6. ERBB2 (Her2, Hs00170433_m1);

7. VIM (Hs00185584_m1);

8. KRT19 (CK19, Hs00761767_s1);

9. TACSTD1 (Ep-CAM, Hs00158980_m1);

10. CD44 (Hs00153304_m1);

11. CD24 (Hs00273561_s1);

12. EGFR (Hs00193306_m1);

13. CRYAB (Hs00157107_m1);

14. FOXA1 (Hs00270129_m1);

15. FOXC1 (Hs00559473_s1).

These TagMan gene expression assays (20×) were pooled together anddiluted with TE buffer to yield 1× Assay mixture. The pre-amplificationwas done in 10 μl volume including 5.0 μl Cells Direct 2× Reaction Mix,0.50 μl 1× pooled Assay Mixture, 1 μl Cell [or human reference RNA(Stratagene, La Jolla, Calif.)], 2.5 μl TE (pH 8.0), and 1 μl RT-Taqenzyme. The RT step was performed at 50° C. for 15 minutes, followed by18 cycles of amplification (95° C. for 15 minutes and then 60° C. for 4minutes). Pre-amplified cDNA were diluted 2 times in TE buffer andstored at −20° C.

CellsDirect™ (from Invitrogen Corp.) qRT-PCR kits deliver highlysensitive and specific, real-time qPCR results directly from cells,without the need for an RNA purification step when testing with lessthan 10,000 cells per reaction to as low as 1 cell. CellsDirect™technology is designed for maximum sensitivity with small samples. Byeliminating costly, time-consuming RNA purification procedures, it isespecially valuable for high-throughput applications and is ideal forgene expression experiments. TaqMan Universal Master Mix (AppliedBiosystems, Foster City, Calif., [TaqMan Universal Master Mix Reagentsprovide a PCR mix that may be used with any appropriately designedprimer and probe to detect any DNA or cDNA sequence].) and 48.48 dynamicarray chips, the NanoFlex™ 4-IFC Controller and the BioMark Real-TimePCR System (Fluidigm Corporation, South San Francisco, Calif.) were usedfor multiplex qRT-PCR. Arrays were performed following the standardFluidigm protocol (Spurgeon et al., “High Throughput Gene ExpressionMeasurement with Real Time PCR in a Microfluidic Dynamic Array,” PLoSONE 3(2): e1662. doi:10.1371 2008). The chip was first primed withKrytox in the NanoFlex™ 4-IFC Controller. Then, 5 μl sample mixturescontaining 2.5 μl 2× TaqMan Universal Master Mix, 0.25 μl DA sampleloading reagent (Fluidigm Corporation, South San Francisco, Calif.), and2.25 μl preamplified cDNA were pipetted into the sample inlets. 5 μlassay mix containing 2.5 μl 20× gene expression assay mix (AppliedBiosystems, Foster City, Calif.) and 2.5 μl DA Assay loading regent(Fluidigm Corporation, South San Francisco, Calif.) were pipetted intothe assay inlets. The chip was then loaded and mixed in the NanoFlex™4-IFC Controller. qRT-PCR reactions of the chip were performed using theBioMark Real-Time PCR System. The cycling program consisted of 10 min at95° C. followed by 40 cycles of 95° C. for 15 sec and 60° C. for 1 min.

Each Biomark chip can assay 48 samples for 48 gene targets. The resultsshown in Table 3 are representative Ct values based on measurements of15 genes in triplicate using only human sequence specific primers, withwater as a negative control. Table 3 below shows a numerical sample ofdata earlier presented as a heat map (not shown). The table indicatesthe profile of expression of three housekeeping genes (GAPDH, Beta Actinand PRLPO) and twelve genes associated with breast cancer in patients Athrough G. While the housekeeping gene levels did not vary from cell tocell, marked differences in gene expression were seen in the other genesmeasured, even from the same patient. Table 4 shows that the values alsovaried from patient to patient.

TABLE 4 FOXA1 CD44 ER PR VIM ERBB2 EpCAM A1 21.4 20.27 24.27 21.55 18.222.63 A2 22.34 B1 27.72 19.05 26.06 B2 B3 23.54 C1 22.99 21.58 D 23.16E1 25.94 24.22 F1 20.75 25.13 F2 G1 23.19 GAPDH B-Actin RPLPO RPLPOCRYAB EGFR A1 14.75 15.95 18.46 18.39 17.12 23.7 A2 19.58 21.03 24.6724.1 B1 19.1 21.91 23.15 23.51 B2 22.62 25.59 26.74 B3 20.92 18.47 23.4122.38 C1 19.88 18.89 23.4 23.66 D 22.51 27.89 25.5 24.77 E1 29.86 32.0527.36 27.79 F1 21.42 22.87 23.55 23.54 F2 21.15 20.12 25.98 27.38 G122.07 24.47 30.09 FOXC1 CD24 CK19 A1 22.63 19.55 17.34 A2 B1 B2 23.43 B324.91 25.21 C1 31.65 D 23.05 E1 F1 24.41 19.5 F2 24.14 26.39 22.29 G1

TABLE 4A Results from Patient A Only GAPDH B-Actin RPLPO CRYAB EGFRFOXA1 CD44 ER A1-1 14.75 15.95 18.46 17.12 23.7 21.4 20.27 24.27 2 14.8116.8 18.33 21.49 22.97 3 14.42 16.67 17.8 23 21.13 4 14.53 15.25 17.8821.71 20.4 5 16.66 18.77 19.85 23.15 6 17.93 19.09 22.54 7 18.09 19.1522.04 25.76 8 13.83 16.38 17.97 20.84 20.13 PR VIM ERBB2 EpCAM FOXC1CD24 CK19 H2O A1-1 21.55 18.2 22.63 22.63 19.55 17.34 2 22.8 20.64 25.0424.17 20.81 18.37 3 18.79 24.65 24.31 19.91 18.67 4 18.42 22.76 5 22.8420.79 25.73 23.41 25.38 19.97 6 22.68 24.72 7 22.85 24.43 25.71 19.66 8

Tables 4 and 4A show gene expression data of 15 genes, where expressionwas performed on single CTCs isolated from seven patients, indicated bythe seven letters A-G. Each of the 15 genes was measured in triplicate(data shown for one measurement) for each single CTC. Patients A, B andF had sequential blood draws, denoted as A1 and A2; B1, B2, and B3; F1and F2. Table 3A shows separate cells from the same patient toillustrate variability among CTCs.

Gene expression profiles for single MCF7 cells spiked into blood andcaptured by the MagSweeper were compared with the parental cell line.There was no marked change in gene expression of the cells afterlabeling and capture. Moreover, at the single cell level, the cellularheterogeneity of the parent cell line was recapitulated in the singlecells captured by the MagSweeper. This is a critical aspect for humanCTC studies: if different populations of CTCs exist in a given cancerpatient, CTC heterogeneity could only be observed at the single celllevel.

Isolation of CTCs from Metastatic Breast Cancer Patients

The above in vitro and in vivo preclinical experiments showed that thepresent device provided gentle isolation and complete purification ofCTCs without perturbing cell viability or gene expression. MagSweeperperformance was then tested in clinical applications. With informedconsent, blood samples were obtained from eleven women with metastaticbreast cancer and five healthy volunteers (normal controls). Patientswith known metastatic breast cancer or healthy normal controls wereconsented prior to sample collection in accordance with Stanford's HumanSubjects Research Compliance Board and HIPAA regulations. Blood wascollected in 10 mL BD Vacutainer plastic EDTA tubes (Becton Dickinson,Franklin Lakes, N.J.). Blood was collected by venipuncture or fromimplanted venous access ports or both. The first 3 cc from each blooddraw was discarded to prevent contamination by skin epithelial cellsfrom the needle puncture site. Then approximately 9 ml of blood wascollected from each human subject and kept at room temperature. Allblood samples were processed within 3 hours of collection. The blood wassplit into three tubes and diluted to 6 ml each with PBS and labeledwith 3.4 μl of Dynabeads® at room temperature with constant mixing for15 minutes. The samples were then placed in an EasySep Magnet (Stem CellTechnologies Vancouver, BC, Canada) for 2 minutes and then mixed foranother 15 minutes. These samples were added to wells of a 6-well plate,brought up to 10 ml each with PBS, and then processed by the MagSweeper.The 4.5 μm beads were coated with the monoclonal BerEP4, which isspecific for an epithelial cell surface epitope (EpCAM) and is furtherdescribed in J Clin Pathol., 1990 March; 43(3): 213-219. The beads wereincubated for 30 min. at room temperature with constant mixing. TheEasySep magnet was used in a premixing step to help attach the beads tothe antibodies.

CTCs were extracted from all eleven (100%) cancer patients and none (0%)of the healthy controls. This is consistent with previous work thatidentified CTCs in 60-100% of patients with metastatic breast cancer and0-1 CTCs in healthy controls. The 235 individual CTCs captured by theMagSweeper were each photographed then analyzed using high throughputmicrofluidic arrays to measure 15-gene expression profiles. For eachsingle CTC, the expression of the following genes was profiled, as shownin FIG. 10: three housekeeping genes (GAPDH, β-Actin and RPLPO); twobreast cancer endocrine biomarkers (estrogen receptor, ER, andprogesterone receptor, PR); two receptor tyrosine kinase genes for whichtargeted therapy is available for breast cancer treatment (ERBB2/HER2and EGFR); three genes previously identified by DNA microarray studiesas being associated with luminal and basal subtypes of breast cancer(FOXA1, FOXC1, and CRYAB); two epithelial genes (CK19 and EpCAM); twogenes for cancer stem cell identification (CD44 and CD24); and amesenchymal gene (vimentin, VIM) implicated in epithelial-to-mesenchymaltransition (EMT) and mesenchymal-to-epithelial transition (MET) andbreast progenitor cells.

All eleven breast cancer patients were receiving chemotherapy at thetime their blood samples were obtained. The MagSweeper used herecaptured CTCs with measurable expression in at least one housekeepinggene in the blood of ten (91%) of the metastatic breast cancer patients.We conservatively defined a CTC as having “robust gene expression” onlyif all three housekeeping genes were expressed; this was done to avoidanalyzing CTCs with degraded RNA, as might be observed in CTCsundergoing apoptosis or cytotoxic degradation. Of the eleven patients,seven (64%) had at least one CTC with robust gene expression. 48 CTCshad robust gene expression. The other 187 CTCs expressed zero to twohousekeeping genes. Consistent with our findings, morphologic andmolecular studies have shown that fragmented or apoptosing CTCs arecommonly observed in the blood of patients undergoing treatment formetastatic disease.

The profiles of standard breast cancer biomarkers (ER, PR, and HER2) forprimary tumor, any available metastases, and CTCs for the seven patientswith robust CTC gene expression data are compared in (Table 5). Therewas considerable discordance between primary tumors and CTCs. Inparticular, among patients who had ER-positive breast cancers excisedyears earlier and who now had multiple distant metastases, their CTCsalmost exclusively showed an ER-negative phenotype. This could explainwhy endocrine therapies ultimately fail to control metastatic disease inpatients with hormone receptor positive primary breast cancer. Allpatients had some CTCs with triple negative (ER negative/PRnegative/HER2 negative) profiles, even though none originally had triplenegative primary tumors. In particular, one patient (Patient B, samplesB1, B2, B3) had a primary breast cancer that showed strong HER2amplification, yet her metastatic disease continued to progress ontrastuzumab, a humanized monoclonal antibody that targets the HER2 cellsurface receptor. Unlike her primary cancer, only one of her CTCsexpressed HER2 and almost all other of her CTCs were triple negative(16/19, 1/1 and 7/7, for samples B1, B2 and B3, respectively). Thetriple negative phenotype of the majority of her CTCs matched the triplenegative phenotype of her biopsied vertebral metastasis, suggesting thatCTC biomarker profiles may better reflect metastatic phenotypes thanprimary tumor profiles. It also may explain trastuzumab resistance inprogressing metastatic disease. In contrast to Patient B, Patient A(sample A1) expressed HER2 in most of her CTCs. Following treatment withtrastuzumab, her CTC count decreased from hundreds to five cells per 9cc of blood (sample A2). Among the five CTCs isolated in sample A2, onlyone expressed all three housekeeping genes and this cell lacked HER2expression.

TABLE 5 CTC Mesenchymal- Partially Differentiated profile like stemdifferentiated tumor Patient Primary Metastatic (ER, PR, Vimentin cellsepithelial epithelial ID tumor(s) tumor HER2) expression (MSC) (PDE)(DTE) A ER−, PR−, N/A Sample 1 (−, −, +) (5/8) 4/8 ----- 1/1 0/8 -----2/8 ----- 1/1 6/8 ----- 0/1 HER2+ (+, −, +) 0/1 (1/8) (−, −, −) (2/8)----- Sample 2 (−, −, −) (1/1) B ER+, PR+, ER−, Sample 1 (−, −, +) 18/19----- 7/19 ----- 8/19 ----- 1/1 ----- 4/7 3/19 ----- 0/1 HER2+ PR−,(1/19) 0/1 ----- 3/7 0/1 ----- ----- 0/7 (left) ER−, HER2− (−, +, −) 0/7PR−, (bone (1/19) (+, −, −) HER2+ metastasis) (1/19) (−, −, −) (right)(16/19) ----- Sample 2 (−, −, −) (1/1) ----- Sample 3 (−, −, −) (7/7) Cunknown ER+, (−, −, −) (3/3) 1/3 0/3 3/3 0/3 PR−, HER2− (bonemetastasis) D ER−, PR+, ER+, (−, −, −) (5/5) 0/5 0/5 2/5 0/5 HER2− PR−,HER2− (lymph node metastasis) E ER+, PR+, N/A (−, −, −) (1/1) 1/1 1/10/1 0/1 HER2 unk F ER+, PR+, ER+, Sample 1 ( −, −, −) (1/1) -- 1/1 -----0/1 0/1 ----- 1/1 ----- 1/1 0/1 ----- 0/1 HER2 unk PR−, --- Sample 2 (−,−, −) 0/1 HER2− (1/1) (chest wall recurrence) G ER+, PR+, N/A (−, −, −)(1/1) 1/1 0/1 1/1 0/1 HER2−

Table 5 above shows the biomarker profiles for seven patients withmetastatic breast cancer who had at least one CTC with robust geneexpression (expression of all three housekeeping genes). Patients A, Band F had sequential blood draws over a four month period. Standardbreast biomarker profiles (ER, PR, HER2) are shown for primary tumor,metastases when available, and CTCs. For standard biomarker profiles andeach CTC phenotype (MSCs, PDEs, and DTEs), the numerator is the numberof CTCs of that phenotype and the denominator is the total number ofCTCs isolated from each sample. Data is shown for only CTCs with robustgene expression. Vimentin was the most commonly observed gene expressedby CTCs. Differentiated tumor epithelial cells (DTEs) were defined asCTCs that expressed ER, PR, HER2, or EGFR and at least one other breastepithelial gene: FOXA1, FOXC1, CRYAB, EpCAM, or CK19. Partiallydifferentiated epithelial cells (PDEs) were defined by lack ofexpression of ER, PR, HER2, or EGFR but still expressed at least oneepithelial marker. We also denoted CTCs to be PDEs if they expressedvimentin alone or CD24 because of where they clustered, although furthercharacterization is necessary. CTCs that co-expressed vimentin and CD44were defined as mesenchymal-like cancer stem cells (MSCs).

Most CTC profiles were striking in their expression of vimentin andCD44. Vimentin, expressed by mesenchymal and breast progenitor cells, isassociated with cell motility, invasion, high tumor grade, andmetastasis). CTCs from six of the seven patients (86%) and 60% of allCTCs with robust gene expression strongly expressed vimentin. Inaddition, we found CD44-expressing CTCs in over 70% of patients and morethan half of all CTCs with robust gene expression. CD44 is a cellsurface adhesion molecule that is expressed by putative breast cancerstem cells. Among cells with degraded profiles (expressing 0-2housekeeping genes), CK19 was the most commonly expressed gene(36%-62%), and vimentin and CD44 were only expressed in 12-27% and0-27%, respectively. This suggests that more epithelial-like cells maybe more susceptible to degradation in the blood stream of patientsundergoing chemotherapy than the more robust vimentin-expressing cells.

In the in vitro experiments, single captured cells recapitulated theheterogeneity of the parental cells. It was thus expected that anyvariation observed in the clinical samples would similarly reflectdistinct cell populations within metastases. When analyzing the isolatedCTCs from patients with metastatic breast cancer, it was found that theydisplayed a spectrum of gene expression, ranging from moredifferentiated tumor epithelial cells (DTEs) to partially differentiatedepithelial cells (PDEs) to mesenchymal-like cancer stem cells (MSCs).DTEs expressed ER, PR, HER2 or EGFR and at least one other breastepithelial marker such as FOXA1, FOXC1, CRYAB, EpCAM, or CK19. PDEslacked expression of ER, PR, HER2 or EGFR, but still expressed at leastone epithelial marker. We also denoted cells to be PDEs if theyexpressed vimentin alone or CD24, although they will require furthercharacterization in a larger series. Finally, we discovered an importantclass of CTCs that co-express vimentin and CD44 without other markers;these cells appear to represent a class of mesenchymal-like cancer stemcells (MSC) or breast progenitor cells. These particular CTCs maycorrespond to the basal B phenotypes described in breast cancer celllines by Neve et al. (R. M. Neve et al., Cancer Cell, 10, 515-527(2006)) and breast progenitor cells identified in primary human breasttumors by Korshing et al. (E. Korsching et al., J. Pathol., 206, 451-457(2005). In two of the three patients with serial blood analyses, CTCpopulations varied over time, showing a decreasing proportion of DTEsamong robustly expressing CTCs. The third patient had only PDEs. We thuspropose that PDEs and MSCs are more chemoresistant than DTEs, and thepresence of these less differentiated CTCs represents a hallmark ofprogressive metastatic disease. The inherent variation of geneexpression of individual CTCs in patients undergoing treatment forwidespread metastatic breast cancer suggests that pooling CTCs forgenetic analysis would obscure specific populations and that single cellanalysis is needed to achieve a complete picture of metastatic biology.In these results 25% of the human CTCs with robust gene expression havea mesenchymal stem cell-like phenotype, which may require specifictherapeutic targeting (R. S. Finn et al., Breast Cancer Res. Treat. 105,319-326 (2007)). Phenotyping individual CTCs in metastatic cancer maylead to more personalized therapy.

Additional Cell Studies

50 HLA-A2 positive cells were spiked as target cells into a solutioncontaining different amounts of HLA-A2 negative cells. The target cells(HLA-A2) were labeled with 4.5 μm magnetic beads functionalized with ananti-HLA-A2 antibody. The capture rate and purity of the targeted cellsisolated by MagSweeper after two rounds of capture-wash-release wasfound to be approximately 60%, independent to the number of backgroundcells. The purity of isolated HLA-A2 cells was 100% until the backgroundcells are in excess of 2×10⁵ and is 89%±2% when the number of backgroundcells is 2×10⁶. In addition, this data indicates the enrichment oftarget cells by 2.5×10⁵-fold when the background cells are as high as2×10⁷.

After visual inspection of the captured cells under fluorescentmicroscopy, we found that the majority of contaminant cells wereattached to magnetic beads. This indicates that the purity is limited bythe antibody specificity and reagent quality.

In another experiment, 50 stained cancer cells from the human breastcancer cell line MCF7 were spiked into 1 ml of stained peripheral blooddrawn from healthy human volunteers. For ease of detection, the targetMCF7 cells and background blood cells were stained with SNARF-1(Invitrogen, CA) and CFDA SE (Invitrogen, CA) fluorescent dyes,respectively, according to the manufacturer's recommendation. The MCF7cells were labeled with 4.5 μm paramagnetic beads functionalized withantibodies against the epithelial cell adhesion molecule (EpCAM) whichis expressed on the cell membrane of epithelial cells, but not onleukocytes or red blood cells. The 4.5 μm magnetic beads permittedisolation of target epithelial cells even with only one bead attached tothe cells, which makes the procedure suitable for isolating CTCs withmoderate to low EpCAM expression.

The capturing efficiency of MCF7 cells by MagS weeper was found to be tobe 66%±10% with the purity of 23%±5%. However, it was found that if theseparation and labeling process was carried out at 4° C., the purity canbe increased to 91%±6%.

To assess whether the MagSweeper protocol perturbs the gene expressionprofile of the CTCs during the isolation process, the genome widetranscriptome expression profile of MCF7 cells was studied usingmicroarray analysis. The expression profiling of 20,000 MCF7 cells grownin culture media, was compared with similar number of MCF7 cellsincubated for thirty minutes with anti-EpCAM magnetic beads before andafter MagSweeper isolation. Coefficients of variation of gene expressionbetween cultured cells and after MagSweeper isolation were comparable,suggesting little evidence of increased variation due to isolation.Moreover, gene expression fold changes between cultured cells andMagSweeper were analyzed. 42% of probe sets have changes less than 10%,another 35% have changes 10-25%, and 17% additional between 25-50%.Statistical analysis of gene expression between the culture cells andMagSweeper reveals that none of the changes are significant at 5% falsediscovery rate (FDR), indicating that the MagSweeper isolation protocoldoes not induce any significant perturbation in the gene expressionprofile of the cells during the isolation process.

Further Manipulation of Isolated Rare Cells

Automated Single Cell Retrieval

The method for obtaining an essentially pure composition containing maycomprise a post-processing sample preparation step that will automatesingle cell identification, extraction, and deposition. This may be doneby automated aspiration and magnetic microextraction, where a singleisolated cell is deposited into a collection tube containing 1-2microliters of fluid in preparation for multiplex molecular analysis.Referring now to FIG. 13, there is shown a schematic illustration of anexemplary fully automated cell extraction system, where one may obtain asingle viable cell from the cluster of bead-coated cells obtained fromthe MagSweeper. In this device, a sample containing targets 1102, i.e.,cell clusters or cells obtained from the collection well of the presentseparation device, are placed on a computer controlled mechanicallydriven stage 1104, which is used to hold the cells. An invertedmicroscope 1106 images the cells and its view is transmitted to a linearCCD image sensor 1108 which can detect cells and their shapes and othervisual characteristics. An interferometer 1110 is also opticallyconnected to the cells to obtain information about the light strikingcells from the illumination source 1112. A control computer 1114receives data from the image sensor 1108 and the interferometer 1110.The computer 1114 also controls an actuator 1116, through a mechanicalpositioned 1118. The actuator 1118 may be connected an automated pipetteor microextractor 1120 or a magnetic device, as described below. Thecomputer uses image recognition software to and information receivedfrom the interferometer and the CCD image sensor. In operation, thesample containing the targets is continuously scanned through a ribbonof illumination. A linear charge-coupled device (CCD) then acquires theimage line by line. The image is then assembled into a two-dimensionalimage in the control computer. The control computer is programmed toidentify each target, as described below. Using stage positioninformation from the interferometer, and line and height informationfrom the CCD, the computer, in response, moves the microextractor tipinto position. The target (individual cell) may then be extracted. Thecontrol computer will then actuate the mechanical positioner and themicroextractor will be moved to the collection tube (not shown) wherethe target is released. This process will be repeated for each target inthe sample.

Automated Pipetting

One method to obtain a single cell from a cell population employs a cellmanipulator system such as the Eppendorf TransferMan NK 2™ can serve asan initial system. However, the TransferMan employs a manually operatedjoystick. A fully automated system may be custom engineered. In oneembodiment, the operator would click a cursor on each target in a liveimage; the combined co-ordinates of the microscope stage and the cursorwould then be fed into a computer that would direct the pipette tip tothe target. The operator would electronically actuate aspiration of thetarget into the pipette. The pipette would then be moved to a collectiontube where the target cell would be dispensed. To create a customsystem, one may set up an off-the-shelf micromanipulator system such asthe Eppendorf TransferMan NK2. This system is attached to the ZeissAxiovert inverted microscope in our laboratory. A motorized stage systemis used to position the samples using operator-assisted visual feedback.Different electronic pipette systems are attached to themicromanipulator for testing. Suitable candidates include handheldinstruments such as a Hamilton Gastight digital syringe hand-heldinstrument that can be set for repetitive aspiration and dispensing,modified for external actuation. The syringes are programmed forextraction and release of a preset volume. Following aspiration of theCTC by either of these electronic pipettes, the pipette can be movedunder joystick control for release into a collection tube. For manualaspiration, we originally used a small pipette tip (150 micron internaldiameter). However, the sharp rim contributed to cell rupture by givingrise to large local stresses that caused shearing of the target, so weswitched to a larger diameter tip (0.5-10 μl micropipette tip, 400micron internal diameter). If cell rupture due pipette shearing createsa problem in the automated pipetting system, one variant of our pipettedesign would be to mount a flat perforated cap across the orifice thatwould provide a cushion. A perforated flat disk silicon end cap can beattached to the end of a glass pipette tip having an end opening of0.25-0.5 mm, with the disk having 3-5 small perforations.

As an alternative to pipetting a single cell, one may use a magneticprobe which can be micromanipulated to attach to a single cell. Therationale for using a localized magnetic field for CTC extraction is toextract the target without excess beads or solution, and to performextraction with potentially less cell rupture than achieved byaspiration. A wire made of a soft magnetic material (e.g., pure ironwhich can be obtained commercially and may be subsequently annealed) tofacilitate CTC release (soft magnets can be easily demagnetized). Toavoid picking up excess beads along the length of the wire, the wirewill be encapsulated by a nonmagnetic material, except at the tip. Withthis configuration, the magnetic force on a target should fall offrapidly. A simple numerical calculation indicates that it should falloff more than 10-fold within a 1-tip radius distance from the tipsurface. The force generated on a target is given by. The forcegenerated on a target is given by

$F = {{VKH}\frac{\partial H}{\partial x}}$where V is the volume of the target, K its magnetic susceptibility and xis distance.

For soft iron, K can exceed 1000 and the maximum field at the surfacecan exceed 1 T (Tesla) but a value of 0.2 T might be a more practicalmaximum. The volume of the target can range from 10⁻¹⁵ to 10⁻¹⁴ m³.Operation of the magnetic microextractor involves bringing the tip intoclose proximity (e.g., less than 100 microns) and then energizing thesolenoid by switching on the current. As before, the microextractor isthen be moved under joystick control to the collection tube. The currentwill be switched off for target release. If the target does not release,one may cyclically reverse the magnetic field with decreasing amplitude.An external magnetic field of larger gradient can also be applied toassist target release, similar to the methods already in use in the laststep of the MagSweeper operation.

Further Details of Construction of the Magnetic Member

FIG. 9 illustrates the effect of the magnetic field on the cells. Inthis embodiment a sleeve 902 tightly fits over the magnetic rod along atleast a portion of the side of the rod 904, and tightly at the tip. (Thespacing is exaggerated for purpose of illustration.) Cells withoutsufficient magnetic labeling 906 inside the boundary layer 908 are onlyaffected in a lateral direction. Cells with beads 910 sufficient to bewithin the magnetic force of the rod are drawn to the rod. Thus it isimportant that the magnetic member used, with sleeve, contain a sharpmagnetic gradient to strongly bind the labeled beads which are in closeproximity to the rod. The relationship is shown in the plot of B² versusdirection z. A steep magnetic gradient will produce a high strengthfield near the magnet surface, in this case the tip. The inserted graphin FIG. 9 illustrates the decay of magnetic flux density B² along the zaxis (away from the rod). Beyond about 6 mm from the tip, it was foundby modeling that there was very little magnetic force, due to the sharpmagnetic gradient.

The present methods may further comprise target recognition technologiesfor CTC identification without user input. Further description of cellimage recognition software may be found in Karacali et al. “Automatedrecognition of cell phenotypes in histology images based on membrane-and nuclei-targeting biomarkers,” BMC Medical Imaging 7:7 (2007), foundonline at www-biomedcentral.com/1471-2342/7/7. As described there, openaccess systems operated by internet web servers, such as EAMUS™, haverecently been developed for extracting quantitative parameters fromimmunohistochemically stained tissue slides and tissue microarrays.Several commercial software packages have also been developed forcytometric analysis of histological slides and tissue microarrays suchas the Tissue Microarray Analysis Software (TMAx) by Beecher Instruments(Beecher Instruments, Inc., 686 Progress Way, Sun Prairie, Wis. 53590,USA), the Extended Slide Wizard by Tripath Imaging (Tripath Imaging,Inc., 780 Plantation Drive, Burlington, N.C. 27215, USA), the DiscoveryImage Analyser by Becton-Dickinson (Becton-Dickinson Biosciences,Postbus 757, 2400 AT Alphen aan den Rijn, The Netherlands). Thesesystems can be trained to identify isolated CTCs with attached beads andantibodies.

Further illustration of magnetic parameters and a possible magneticmember configuration is set forth in FIG. 14. As shown there, one mayuse a magnet 1402, which need not be a rod, but may be extended in widthtransverse to the polar direction. It may be swept through the poollaterally. The magnet 1402 comprises at one pole a tapered region 1404which may be hemispheric, or, alternatively, as shown, comprise acompound linear narrowing, having region of first narrowing 1406 and adistal region of increased narrowing 1408, ending in a sharp tip or atip having a small radius of curvature at a pole, e.g., the South pole.The magnetic field, as illustrated by magnetic lines of force at 1410,is close to the non-magnetic floor of the container 1416. Theillustrated configuration improves the magnetic gradient over a simpleblunt end or hemispherical end of a magnet. This configuration can beapplied to the tips of rods, to multiple magnets, and magnets withopposing poles can be arrayed so that the gradients are steeper.

Culture of an Isolated Cell

In addition to conducting genetic analyses on isolated individual cells,the present device and methods may be used to capture viable cells thatcan be grown in culture. Models for testing viability of captured cellswere developed by generating tumor xenografts from both human breastcancer cell lines (MDA-MB-231) and primary tumors excised from breastcancer patients and orthotopically implanted into the mammary fat padsof immunocompromised female mice (NOD-SCID). MDA-MB-231 cancer cellsformed mammary fat pad tumors and macroscopic lung metastases in fortymice. Mouse blood was taken and hundreds of CTCs were captured usinghuman-specific EpCAM-labeled magnetic beads. These CTCs were placed intoculture media and incubated. The CTCs attached to the culture dishes andrapidly proliferated. Control mice injected with saline did not developprimary tumors or metastases, and their blood contained no CTCs. Also,three tumor xenografts were generated from primary human breast cancers.Tumor tissue was excised from female patients and implanted into mousemammary fat pads. Human CTCs were isolated from mouse blood of all threexenograft models and CTCs from one of the models was maintained inculture. To our knowledge, this is the first demonstration of isolationof viable human CTCs from primary human tumor xenografts. These methodspave the way for culturing patient-specific CTCs that may be used fordrug testing, drug discovery, or other studies.

The foregoing shows that the present device is able to isolate viableand pure circulating tumor cells from whole blood for genetic analysis.The device and present methods may also be applied to capture of fetalcells from maternal blood. Capture of spiked cells and tumor cells showno contaminating blood cells, and we are working on methods to improvecapture rates, which currently average 25%. Here, we compare multiplexqRT-PCR data of single CTCs isolated from two patients with metastaticbreast cancer with the phenotype of primary tumors and metastases.In-patient SM014, although her primary tumor was ER−/PR−/HER2+, the CTCwas ER−/PR−/HER2−, which matched the phenotype of her bone metastaticdisease that progressed on trastuzumab. In-patient SUBL017, the primarytumor was ER−/PR+/HER2− and an intervening supraclavicular LN metastasiswas ER+/PR−/HER−. The CTCs we captured and analyzed from this patient,now with progressing bone metastases of unknown receptor status, displayan ER−/PR−/HER2− phenotype. We have also found that gene expression ofCTCs from the same patient vary. Our data indicate that single CTC geneexpression may be highly variable and may reflect the heterogeneity ofcells from single or multiple metastatic lesions.

The use of a robot allows the device to be programmed to utilizemultiple samples, such as in a 6 well plate. The robot is programmableto vary capture and release configurations for optimizing captureefficiency and sample purity. Robot works with all commerciallyavailable immunomagnetic particles. Modifications can be made to therobot, such as allowing the magnetic rods with plastic sleeve to move totheir next station (capture, wash, release station) in a circular (byputting the wells on a conveyor belt-type apparatus), rather than thelinear rod-arm advancement as is currently configured. We may also addan orthogonal arm for manual or robotic sliding of an additionalmagnetic plate under the final release station to facilitate cellrelease from the plastic sleeve.

The present device may, in certain aspects, be characterized ascomprising, firstly, magnetic rods that sweep through blood or othersolution. These rods are actual magnets (axial or diametric) rather thanmetals magnetized by an external magnet. Secondly, it comprisesremovable plastic sleeves over magnets to facilitate multiple releasecycles to assure high capture efficiency and high purity (nocontaminating cells—other devices have a 100-1000:1 ratio ofcontaminating cells to captured cells). Thirdly, it comprises anactuator for sweeping the magnets and for carrying out a sweeping orshaking step at the end of release cycle to assure release of capturedcells into appropriate solution. Fourthly, it uses plastic (for a sleeveover the magnet) that has lowest non-specific binding of contaminatingcells. Fifthly, it has the ability to capture rare cells at very lowinput numbers. For example, cancer blood samples may have 0-10 cells per7.5 cc tube.

In the present method, one may obtain a composition having cells coveredwith numerous beads, and which contain a significant number (at least25%) of target cells. Target cells may be picked by hand through amicroscope and a micropipette, and the DNA, RNA or other materialextracted, or the cell may be cultured.

CONCLUSION

The above specific description is meant to exemplify and illustrate theinvention and should not be seen as limiting the scope of the invention,which is defined by the literal and equivalent scope of the appendedclaims. Any patents or publications mentioned in this specification areindicative of levels of those skilled in the art to which the patent orpublication pertains as of its date and are intended to convey detailsof the invention which may not be explicitly set out but which would beunderstood by workers in the field. Each and every patent or publicationwhich is cited for further information is intended to be and hereby isincorporated by reference to the same extent as if each was specificallyand individually incorporated by reference, to the fullest extentpermitted by law.

REFERENCES

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What is claimed is:
 1. A device for capture and isolation of targetcells from a mixed cell population wherein the target cells are labeledwith magnetically responsive material to form labeled target cells,comprising: (a) a container for holding the labeled target cells; (b) amagnetic member having a tip end and another end connected to anactuator; (c) a sleeve between the magnetic member and the mixed cellpopulation to prevent direct contact of cells with the magnetic member,said sleeve being nonmagnetic and nonadherent to cells in the mixed cellpopulation; (d) said sleeve being separable from the magnetic member bythe actuator, which actuator causes movement between the magnetic memberand the sleeve, whereby the magnetic member is moved more or less intothe sleeve; and (e) said actuator being constructed and arranged forcausing in the device (i) relative orbital stirring movement between themagnetic member and the mixed cell population to contact the magneticmember with a majority of cells in the mixed cell population, and (ii)movement of the magnetic member into and out of the container toretrieve target cells from the mixed cell population.
 2. The device ofclaim 1 wherein the magnetic member is a rod having a diameter of atleast 4 mm to produce a high magnetic field strength and having atapered tip for producing a high magnetic gradient.
 3. The device ofclaim 1 where the magnetic member has a tapered tip.
 4. The device ofclaim 1 wherein the magnetic member possesses a field strength at thetip of at least 0.5 Tesla.
 5. The device of claim 4 where the magneticmember is a rare earth magnet.
 6. The device of claim 4 where themagnetic member has a pull strength of at least 70 pounds.
 7. The deviceof claim 4 where the magnetic member has a surface field strength of 0.2to 1 Tesla.
 8. The device of claim 1 where the magnetic member comprisesneodymium, iron and boron.
 9. The device of claim 1 where the sleeveconsists essentially of a material selected from the group consisting ofvinyl polymer, paramagnetic metal, ceramic material, and polyHEMA. 10.The device of claim 1 where the magnetic member consists essentially ofneodymium, iron and boron alloy.
 11. The device of claim 1 furthercomprising one or more containers for holding the sample, for holding awash solution and for holding target cells after isolation from themixed cell population.
 12. The device of claim 11 where there areseparate containers for holding sample, wash solution and target cells.13. The device of claim 11 where the containers comprise a materialwhich is non-adherent to target cells.
 14. A device for capture andisolation of target cells in a sample having a mixed cell population oftarget cells and contaminant cells, wherein the target cells are labeledwith magnetically responsive material to form labeled target cells,comprising: (a) a container for holding the sample; (b) a magneticmember having a tip; (c) a sleeve between the magnetic member and themixed cell population to prevent direct contact of cells with themagnetic member, said sleeve being nonmagnetic and nonadherent to cellsin the mixed cell population; (d) said sleeve being separable from themagnetic member by an actuator that causes movement between the magneticmember and the sleeve whereby the magnetic member is moved more or lessinto the sleeve; and (e) said actuator being constructed and arrangedfor causing (i) relative stifling movement applied in concentric circlesin the container between the magnetic member and the mixed cellpopulation to contact the magnetic member with a majority of cells inthe mixed cell population, and (ii) movement of the magnetic member intoand out of the container to retrieve target cells from the mixed cellpopulation.
 15. The device of claim 14 further comprising a containerwhich comprises a portion having a magnet opposite the magnetic member,for attracting isolated labeled target cells from the magnetic member informing an isolated population.
 16. The device of claim 14 wherein thecontainer defines an open pool for holding the sample.
 17. The device ofclaim 14 comprising only one magnetic member in one container.
 18. Thedevice of claim 14 comprising multiple magnetic members controlled by asingle actuator.
 19. The device of claim 14 further comprising a probefor extracting a single target cell from a collection of target cellsisolated after removal of contaminant cells.
 20. The device of claim 19comprising a computer programmed with image recognition software andcontrolling the probe.
 21. The device of claim 14 further comprising amagnet for removing target cells from the sleeve.